KR101437381B1 - Method for triploid verification of Pacific oyster - Google Patents
Method for triploid verification of Pacific oyster Download PDFInfo
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Abstract
Description
The present invention relates to a method for discriminating triploids, and more particularly, to a gene kit for triplet discrimination.
The economic utility of polyploidy has led to the development of triploids, which are used in a variety of agricultural systems, and these studies have been particularly active in bivalve molluscs (Beaumont, AR, and JE Fairbrother, J. Shellfish Res. , 10: 1-18, 1991; Nell, JA, Aquaculture 210: 69-88, 2002; Piferrer, F. et al ., Aquaculture 293: 125-156, 2009). Marc Biotechnol ., 7: 318-330, 1996), and the production of the mulberry oysters (Guo & Allen, Biol. Bull . 187: 309-318,1994; Guo et al ., Aquaculture 142: 149-161,1996; McCombie et al . 2005) contributed greatly to the triamc oyster industry obtained by crossing tetraploid and diploid oysters (Guo et al ., Aquaculture 142: 149-161, 1996). Triploid X diploid hybridization can provide highly productive triploids, which can lead to an overall expansion of triploid oysters in the shellfish industry and natural ecology.
In mollusks, triploids are known to be induced by the inhibition of first or second polar body formation by heat, pressure or chemical shock (Beaumont, AR, and JE Fairbrother, J. Shellfish Res. , 10: 1-18, 1991). Continued studies have reported the formation of tetraploids of Pacific oyster (Crassostera giga) through the study of Guo and Allen, in which cytochalasin B was treated to produce triploid egg egg, inhibited the formation of tetraploids by inhibiting the formation of the first polar body. Although the resulting breeding ability of the triploid was reduced, the triplet was able to produce female germ cells. In addition, studies have been continuing to develop a technology for producing oval oysters in order to overcome the problems of supply restriction caused by the patents for producing oval oysters. Therefore, it is required to develop a method for determining the correct level of drainage after inducing drainage in an individual, or for detecting the drainage of an object in an oyster culture habitat environment.
Have been used in the past to identify polymorphisms using a variety of methods (e.g., cell analyzers, karyotyping and particle size analysis). Flow cytometry, which compares the polymorphism level and DNA content of the cells to the control, has been usefully applied to triploid discrimination, since multiple samples can be analyzed simultaneously and the relative proportion of induced triploids can be analyzed (Harrell, RM meat al ., Aquaculture 137: 159-160, 1995). However, this method is also about to predict the polymorphism level, and it takes a lot of time and money to measure the level of intracellular DNA, and it takes a lot of labor to perform the actual operation. Analysis of red cell size and chromosomal analysis can provide the most accurate information on polymorphic levels, but these also have time and labor intensive problems. As a method of determining the other polymorphic levels, the method of imaging analysis (G ㅹ rard, A. et al ., Aquacult . Fish Manage , 25: 697-708, 1994)), microfluorometry (Durand, P. et al ., Nippon Suisan Gakkaishi 56: 1423- 1425, 1990) and nuclear sizing (Gardner et al ., J. Shellfish Res . 15: 609-615,1996).
In addition, polymorphic DNA markers, such as microsatellite, have been used (Slabbert et al ., African Journal of Marine Science 32 (2): 259-264, 2010). Microsatellites or simple sequence repeats (SSRs) have polymorphisms, multiple alleles, homology, and regeneration. They are used to identify genetic diversity in individuals and to identify molecular markers (Chambers, GK et al ., Comparative Biochemistry and Physiology Part B 126: 455-476,2000). The utility of the molecular marker is based on the heterozygosity locus present in the female and the gene locus inherited from males of different size from the two female alleles. A variety of gene loci can be more usefully applied to discriminate triploid characteristics of an individual (Magoulas, A. et al., Animal Genetics, 29: 69-70, 1998; Li, G. et al ., Molecular Ecology Notes , 3: 228-232, 2003; Sekino, M. et al., Mar . Biotechnol ., 5: 227-233, 2003).
SUMMARY OF THE INVENTION The present invention has been made to solve the above-mentioned problems, and it is an object of the present invention to provide a method for effectively discriminating drainage of oysters. However, these problems are exemplary and do not limit the scope of the present invention.
According to one aspect of the present invention, there is provided a multiplex kit for oyster mobility discrimination comprising three or more primer sets selected from the group consisting of the following primer sets 1 to 6:
A primer set 1 consisting of a forward primer having a nucleic acid sequence represented by SEQ ID NO: 1 and a reverse primer represented by SEQ ID NO: 2;
A primer set 2 consisting of a forward primer having a nucleic acid sequence represented by SEQ ID NO: 5 and a reverse primer having a nucleic acid sequence represented by SEQ ID NO: 6;
A primer set 3 consisting of a forward primer having a nucleic acid sequence represented by SEQ ID NO: 7 and a reverse primer having a nucleic acid sequence represented by SEQ ID NO: 8;
A primer set 4 consisting of a forward primer having a nucleic acid sequence represented by SEQ ID NO: 15 and a reverse primer having a nucleic acid sequence represented by SEQ ID NO: 16;
A primer set 5 consisting of a forward primer having a nucleic acid sequence represented by SEQ ID NO: 19 and a reverse primer having a nucleic acid sequence represented by SEQ ID NO: 20; And
A primer set consisting of a forward primer having a nucleic acid sequence represented by SEQ ID NO: 21 and a reverse primer having a nucleic acid sequence represented by SEQ ID NO: 22;
The kit may comprise the primer sets 1, 2 and 3 (panel 1).
The kit may comprise the primer sets 4, 5 and 6 (panel 2).
The kit may include all of the primer sets 1 to 6 (panel 3).
According to another aspect of the present invention, there is provided a DNA amplification method comprising amplifying genomic DNA of oyster with the kit; An electrophoresis step of electrophoresing the amplified DNA fragment; And statistically analyzing the band pattern of the electrophorpted DNA fragment.
The present inventors used a number of gene loci and combinations thereof to develop molecular markers for ploidy discrimination. Three primers can increase economic efficiency and yield per data per reaction and are applied to multiplex PCR techniques to obtain results more economically and faster than single gene locus PCR reactions. By increasing the types of primers included in the multiplex, the accuracy of the analysis could be increased, but also if the sizes of the PCR products obtained from the various gene loci are similar, the inaccuracy can also be increased. Therefore, the number of primers included in the multiplex was selected so as to obtain reliable results with a minimum number of primer pairs. (1), (2) and (3), panel 2 (primer sets 4, 5 and 6), or panel 3 (including primer sets 1 to 6) And thus can be applied to a variety of applications.
According to an embodiment of the present invention as described above, a multiplex kit for oyster drainability discrimination can be implemented. Of course, the scope of the present invention is not limited by these effects.
1 is a graph showing electrophoresis results showing alleles of six gene loci contained in a multiplex kit (panel 1, panel 2 and panel 3) according to an embodiment of the present invention.
Definition of Terms:
The terms used in this document are defined below.
It is known that in the case of three-folded shellfish such as oysters, it is increased to about 30% to 40% or 50% to 70% depending on the species. In this case, it is known that the quality of fish paste improves and shows excellent growth (Guo, X., J. Shellfish Res . , 18: 266-267, 1999; Guo. X. et al ., Genetics 138: 1199-1206, 1994). Due to the gigantism of this triplet, triploid studies on molluscs of various aquatic organisms are being conducted. Although several trials have been conducted on several oysters ( C. virginica , Saccostrea glomerata , and Ostrea Edulis ), only Pacific oyster triploids have been commercially used, where the triangular pyramids are tetraploid and diploid ) (Guo, X., et . ≪ RTI ID = 0.0 > al ., Aquaculture 142: 149-161, 1996; Nell, JA, Aquaculture , 210: 69-88, 2002). However, the production of quadriceps oysters was not applicable to all species of oysters, and the production of quadrice oysters is also known to be difficult for some species, such as the Sydney rock oyster (Nell, JA, Aquaculture , 210: 69-88, 2002 ). Therefore, it is expected that the method according to one embodiment of the present invention can be applied to the development of a trichome and a mollusk.
SNP: Abbreviation for single-nucleotide polymorphism, which means that there is a change in the base of a certain locus between alleles within a population or within a population.
MS: Abbreviation of microsatellite, sometimes referred to as simple sequence repeat (SSR) or short tandem repeat (STR), in which two to six base pairs are repeated at a particular locus in the genome It means the genome structure, and the number of repetition often varies between individuals or alleles, and is used for individual identification or population genetics analysis.
Haplotype: A specific combination of alleles (or DNA sequences) in adjacent loci on a single chromosome, often haplotypes between individuals or between alleles. do.
FST: Fixation index, a measure of population differences due to genetic structure, predicted from genetic polymorphism data such as single-nucleotide polymorphism (SNP) or microsatellite (MS) And is an indicator that is typically used statistically in population metastasis.
Na: number of alleles per locus (number of alleles per locus)
AR: allelic richness is the average number of alleles per locus in a population.
H e : expected heterozygosity, which represents the degree of heterozygosity predicted in the target masturbation within the population, and for a population of diploid individuals is calculated as:
Where m is the number of alleles in the target mood and f i is the allele frequency of the ith allele.
Ho is the observed heterozygosity, which is the value of the observed allele divided by the total genotype, and for a population of diploid individuals is calculated as:
Where n is the number of individuals in the population, and a i1 and a i2 are alleles of the individual I in the target masturbation.
FIS: The inbreeding coefficient, which is the ratio of the variation in subpopulation within an individual. The higher the FIS, the greater the degree of inbreeding.
DETAILED DESCRIPTION OF THE INVENTION [
Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples. It should be understood, however, that the invention is not limited to the disclosed embodiments and examples, but may be embodied in many different forms and should not be construed as being limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, It is provided to fully inform the owner of the scope of the invention.
Example 1: Acquisition of oyster samples
Triploid oysters (n = 30) were collected from the oyster farms located on the west coast of Korea, and diploid oysters (n = 96) were collected from the west and southern coastal waters of Korea. The mantle tissue was stored in a 100% ethanol solution immediately after harvesting, and the DNA was extracted from the tissue after transferring to the laboratory. The whole DNA was separated for each sample using a MagExtractor MFX-6100 automated DNA extraction system (Toyobo, Osaka, Japan), and the extracted genomic DNA was analyzed using a Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Barrington, IL, USA) and stored at -20 ° C until analysis of microsatellite DNA.
Example 2: Primary genotype test
A total of 30 microsatellite markers were selected based on the number of alleles and the annealing temperature (Magoulas, A., et al., Animal Genetics, 29: 69-70, 1998; Li, G., et al ., Molecular Ecology Notes , 3: 228-232, 2003). Eleven markers were selected and used to produce a PCR product identified as not null allele among the selected markers (see Table 1).
PCR amplification was performed using 0.25 U Ex taq DNA polymerase (TaKaRa Biomedical Inc., Shiga, Japan), 1 x PCR buffer, 0.2 mM dNTP mix, 10 pmol forward or reverse primer (5 ' -Terminal was labeled with 6-FAM, NED and HEX dyes, which were performed using PE Applied Biosystems (Boston)) and 10 μL reaction solution containing 100 ng template DNA and PTC 200 DNA Engine (MJ Research, Waltham, Mass., USA). The PCR reaction conditions were as follows: 95 ° C for 11 minutes and 35 cycles (1 minute at 94 ° C, 1 minute at annealing temperature, 1 minute at 72 ° C, described in Table 1), extension 72 ° C for 5 minutes.
Analysis of microsatellite gene polymorphisms was performed using ABI PRISM 3130 XL automated DNA sequencer (Applied Biosystems, Boston) and the allele was determined according to the relative PCR product size relative to the molecular site marker (GENESCAN 400 HD [ROX]; PE Applied Biosystems, Boston).
In the above Table 1, AT means annealing temperature, Na means number of alleles per locus, Ho means observed heterozygosity, , And He means expected heterozygosity.
Example 3: Preparation of multiplex PCR
After performing the first amplification in Example 2, six polynucleotide dinucleotide microsatellites were selected on the basis of observed heterozygosity (Ho) and PCR products in order to ensure separation of microsatellite genes And multiplex PCR have been classified into two groups. Primer sets that are easily labeled using general fluorescent dyes (eg, NED, HEX, and FAM) , And thus panel 1, 2 or 3 was prepared.
Panel 1 was prepared to contain ucdCg170, Cg108, and Cg49 loci to produce PCR products of 84 bp to 201 bp in size. Panel 2 contained ucdCg129, ucdCg186, and ucdCg189, which produce PCR products of 204 bp to 326 bp in size. , And ucdCg151 loci (loci). In addition, a panel 3 containing primers for the six gene loci contained in the panels 1 and 2 was additionally constructed.
The forward primers for each gene locus were labeled with different dyes. ucdCg170 and ucdCg129 were labeled with NED, ucdCg186 was labeled with HEX, and Cg49 and ucdCg151 were labeled with FAM (Applied Biosystems, Foster City, CA, USA). The annealing temperature and primer concentration were optimized for each panel, so that the peak of each primer set was similar at the time of amplification. This is summarized in Table 2 (see Table 2).
Experimental Example 1: Analysis of oyster drainage using multiplex PCR
In order to detect triploids of C. gigas quickly and economically, microsatellite DNA markers were used conventionally. The method can be used to distinguish trimeshed oysters from triploid ovine production and natural or artificial cultivation sites. In previous studies, 11 microsatellite markers in 384 diploid individuals have been reported to exhibit polymorphism (Magoulas et al ., Animal Genetics , 29: 69-70, 1998; Li et al ., Molecular Ecology Notes 3: 228-232, 2003). Similarly, three alleles have been reported for all three loci in triplicate.
Eleven microsatellite gene markers were selected, primers were prepared in Examples 2 to 3, and 6 primer sets were selected based on the annealing temperature and the heterozygosity value expected in the range of 0.71 to 0.86 , And a multiplex kit containing the same was prepared. At this time, the heterozygosity value of the primer set is very similar to that shown by the study of Slabbert et al. ( Afr. J. Mar. Sci . 32: 259-264, 2010).
For the 30 oyster cultivars obtained from the oyster farm in Example 1, it was judged whether or not it was a triploid using the multiplex PCR kit of Example 3. The results of the electrophoretic analysis of the genotypes using the panels 1 and 2 according to one embodiment of the present invention for the diploid and triple individuals are shown in FIG. In order to reaffirm the results obtained using multiplex PCR according to one embodiment of the present invention, all genotypes were compared to the bands obtained through a single PCR technique and as a result the same results were obtained in all test samples.
As a result of analysis of 30 individuals, it was found that there were 3 alleles in at least 2 loci, 23 (76.7%) in panel 1 and 16 (53.3%) in panel 2 Respectively. In addition, panel 3 containing all of the primer sets of panels 1 and 2 showed 3 alleles at 6 gene loci in only one sample out of 30 individuals, with 13 individuals (43.3% ) Have three alleles at three loci (see Table 2).
The multiplexing panel (panel 1, panel 2) including three primer pairs according to one embodiment of the present invention can be used to discriminate 100% of the drainage of the oyster. The panel 1 and panel 2 were each composed of three primer sets, and the PCR products generated using the primer set showed different sizes. Polymorphism can be sufficiently discriminated by using only one gene locus having three alleles, so that it is considered that any one of the two panels can be sufficiently used for discriminating the drainage ability. Panel 2 can also be used to reconfirm the incorrect results.
In addition, even if the primers included in the panel 1 and the panel 2 are labeled with the same dye, the panel 3 containing all of the primer sets can be used because it produces PCR products that do not overlap. The panel 3 is expected to be usefully applied because it not only saves time and money in polymorphism discrimination, but also has accuracy.
In addition, the above results demonstrate that micro-gene markers can be usefully used to determine the ploidy of each individual. It is expected that the determination of drainage of each individual can be applied quickly and with repeatability, and it can be utilized in drainage studies. It is also expected that this method can be used to determine whether or not the transition from a triploid state to a diploid state occurs during the development of ovules.
While the present invention has been described with reference to exemplary embodiments, it is to be understood that the invention is not limited to the disclosed exemplary embodiments, but, on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the invention. Accordingly, the true scope of the present invention should be determined by the technical idea of the appended claims.
<110> Republic of Korea (National Fisheries Research and Development Institute) <120> Method for triploid verification of Pacific oyster <130> PD13-0809 <160> 22 <170> Kopatentin 2.0 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <220> Cg108 forward primer (primer set 1) <400> 1 atatgtaatg attacgaaac tc 22 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> Cg108 reverse primer (primer set 1) <400> 2 gtatgagatt tggttccacc 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CGE009 forward primer <400> 3 ttcgttgaag gtgacaagtg 20 <210> 4 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> CGE009 reverse primer <400> 4 gcattttggg atgaacaga 19 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> UcdCg170 forward primer (primer set 2) <400> 5 tggtggtcag tgaatgtgag a 21 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> UcdCg170 reverse primer (primer set 2) <400> 6 cggacagtag ccttttaaca ca 22 <210> 7 <211> 23 <212> DNA <213> Artificial Sequence <220> Cg49 forward primer (primer set 3) <400> 7 atcaggggta aattaaagta agc 23 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> Cg49 reverse primer (primer set 3) <400> 8 ccacagacga tttcatatat cctg 24 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> ucdCg109 forward primer <400> 9 gctatggttg tcatcctcga a 21 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ucdCg109 reverse primer <400> 10 tgcctttatc ggttttgctt 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ucdCg198 forward primer <400> 11 gaaagacacg accggagaga 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ucdCg198 reverse primer <400> 12 ctgatgatgt cccacacctg 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ucdCg181 forward primer <400> 13 caccccaaag gaccacatac 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ucdCg181 reverse primer <400> 14 tgtcagcatg ggtaagtcca 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> The ucdCg129 forward primer (primer set 4) <400> 15 cgaatttttc ggacatcgtt 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> UcdCg129 reverse primer (primer set 4) <400> 16 gtggtatgcc tgcatcatgt 20 <210> 17 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> CGE005 forward primer <400> 17 aaagatgaat ggttgggag 19 <210> 18 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> CGE005 reverse primer <400> 18 aattaaggaa aacggatgc 19 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> UidCG186 forward primer (primer set 5) <400> 19 gccgccgatt ctcttagatt 20 <210> 20 <211> 22 <212> DNA <213> Artificial Sequence <220> UcdCg186 reverse primer (primer set 5) <400> 20 gggctagcta gtcatcaccc ta 22 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> The ucdCg151 forward primer (primer set 6) <400> 21 aggtaatccg caaaccagtg 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> UcdCg151 reverse primer (primer set 6) <400> 22 gcattgcgtc aggattaggt 20
Claims (5)
A primer set 1 consisting of a forward primer having a nucleic acid sequence represented by SEQ ID NO: 1 and a reverse primer represented by SEQ ID NO: 2;
A primer set 2 consisting of a forward primer having a nucleic acid sequence represented by SEQ ID NO: 5 and a reverse primer having a nucleic acid sequence represented by SEQ ID NO: 6;
A primer set 3 consisting of a forward primer having a nucleic acid sequence represented by SEQ ID NO: 7 and a reverse primer having a nucleic acid sequence represented by SEQ ID NO: 8;
A primer set 4 consisting of a forward primer having a nucleic acid sequence represented by SEQ ID NO: 15 and a reverse primer having a nucleic acid sequence represented by SEQ ID NO: 16;
A primer set 5 consisting of a forward primer having a nucleic acid sequence represented by SEQ ID NO: 19 and a reverse primer having a nucleic acid sequence represented by SEQ ID NO: 20; And
A primer set consisting of a forward primer having a nucleic acid sequence represented by SEQ ID NO: 21 and a reverse primer having a nucleic acid sequence represented by SEQ ID NO: 22;
An electrophoresis step of electrophoresing the amplified DNA fragment; And
And statistically analyzing the band pattern of the electrophoresed DNA fragment.
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| KR102311743B1 (en) * | 2020-12-10 | 2021-10-12 | 대한민국 | Identification method of triploid abalone, Haliotis discus hannai using genetic marker |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20110007666A (en) * | 2009-07-17 | 2011-01-25 | 한국해양연구원 | Identifying method of marine species, polynucleotide probe, dna chip and kit for identifying the same |
| KR20120013512A (en) * | 2010-08-05 | 2012-02-15 | 주식회사 씨에버 | Method of producing a viable tetraploid oyster |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20110007666A (en) * | 2009-07-17 | 2011-01-25 | 한국해양연구원 | Identifying method of marine species, polynucleotide probe, dna chip and kit for identifying the same |
| KR20120013512A (en) * | 2010-08-05 | 2012-02-15 | 주식회사 씨에버 | Method of producing a viable tetraploid oyster |
Non-Patent Citations (2)
| Title |
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| Aquaculture, Vol. 288, pp. 172-183 (2009.03.20.) * |
| MARINE BIOTECHNOLOGY, 2005.05.05 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| KR102311743B1 (en) * | 2020-12-10 | 2021-10-12 | 대한민국 | Identification method of triploid abalone, Haliotis discus hannai using genetic marker |
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