JP2010124797A - Method for discriminating edwardsiellasis sensitivity of flatfish - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for discriminating the edwardsiellasis of flatfish, enabling flatfish to be efficiently bred through discriminating flatfish tending to suffer from edwardsiellasis using a specific gene marker. <P>SOLUTION: The method includes the following process: primers are used, among 5'-TCT AGT GTT TGA GCA GAT AGG AAG GG-3' (Sequence No.1), an oligonucleotide composed of consecutive at least 10 bases, and among 5'-CTC TTA GTG GCA CTC CAT CAC ACT G-3' (Sequence No.2), an oligonucleotide composed of consecutive at least 10 bases, a nucleic acid amplification reaction process with a flatfish-derived nucleic acid as template is conducted, and the resultant amplification product is analyzed. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for discriminating the susceptibility of Japanese flounder to Edwardjeellosis using a DNA marker.

Edovagerosis is a typical disease of cultured Japanese flounder caused by infection with Edwardla thalda, a type of intestinal bacterium, and is likely to occur at high water temperatures and has a high mortality rate. The damage caused by edwardiasis in flounder culture is estimated to reach about 300 million yen per year.
Conventionally, inactivated vaccines have been developed that are effective against edwellerosis in fish such as Japanese flounder and Thailand (see Patent Document 1 and Patent Document 2).
Patent Document 3 describes a method for determining the resistance of Japanese flounder lymphocystis disease using a specific gene marker.
However, no method has yet been developed to genetically determine flounder susceptibility to edovagerosis.

JP 2008-50300 A JP 2007-238505 A JP 2004-81155 A

  The problem to be solved by the present invention is to identify a flounder that is susceptible to edegelosis using a specific genetic marker, and to determine the susceptibility of flounder to edewellerosis that enables efficient aquaculture without such flounder. It is to provide.

The present applicant has clarified two strains (disease resistant strain: KP-C and susceptible strain: KP-B) having different susceptibility to edovagerosis in flounder culture that has been continued for many years.
And the hybrid first generation (F ₁ : KP-CB) was produced from these two strains having different susceptibility to edwardiasis. Thereafter, one-to-one mating was performed using F ₁ (KP-CB) to produce a second hybrid (F ₂ : KP-CBCB).
QTL analysis using DNA markers was performed on the produced F ₂ (KP-CBCB), F ₁ (KP-CB) and their parents (KP-C, KP-B).

Usually, in applying the analysis model by F ₂ is the parent fish KP-B, KP-C is BB, homozygotes marker type for _{CC, F 1 (KP-CB} ) heterojunction all CB F ₂ (KP-CBCB) produced by crossing the body and further F ₁ is expected to appear at a ratio of BB: CB: CC of 1: 2: 1. Therefore, samples of heterozygote CB are excluded, and only BB and CC homozygotes are compared and analyzed.
As a result of the analysis by F ₂ , the inventors have found the gene linkage map {M. M.M. Coimbra, Tokyo Fisheries University Doctoral Dissertation (2001); Aquaculture (2003)}, and as a marker locus involved in the susceptibility of flounder edwellerosis, the gene linkage group (LG) 4 in this flounder gene linkage map Marker locus Poli1506TUF was revealed.

It was found that, at the marker locus (linkage group 4: Poli 1506 TUF) involved in the susceptibility of flounder edwellerosis, the mortality rate increased when the marker locus (allele) derived from the sensitive line: KP-B became a homozygote.
Then, using a specific primer related to LG4 marker locus Poli1506TUF, a nucleic acid amplification reaction method using a flounder-derived nucleic acid as a template is performed, and the obtained amplification product is analyzed, thereby discriminating individuals susceptible to Edwardagerosis. be able to.

  The method for determining the susceptibility of Japanese flounder to Edwardjeellosis according to the present invention includes an oligonucleotide part consisting of at least 10 consecutive bases in 5′-TCT AGT GTT TGA GCA GAT AGG AAG GG-3 ′ (SEQ ID NO: 1), and 5′-CTC TTA GTG GCA CTC CAT CAC ACT G-3 ′ (SEQ ID NO: 2), using as a primer an oligonucleotide part consisting of at least 10 consecutive bases, a nucleic acid amplification reaction using a flounder-derived nucleic acid as a template The flounder susceptibility to edovagerosis is determined by analyzing the amplification product obtained by the method.

  For example, among 5′-TCT AGT GTT TGA GCA GAT AGG AAG GG-3 ′ (SEQ ID NO: 1), an oligonucleotide portion consisting of at least 10 consecutive bases, and 5′-CTC TTA GTG GCA CTC CAT CAC Using ACT G-3 ′ (SEQ ID NO: 2), the oligonucleotide chain consisting of at least 10 consecutive bases as a primer, the polymerase chain reaction method is performed on the flounder genomic DNA, and the resulting product is subjected to gel electrophoresis To confirm that there are bands at 148 bp and 151 bp.

According to the present invention, since flounder that is sensitive to edovagerosis can be discriminated, by eliminating such flounder, it is possible to suppress the mortality due to edovagerosis in the culture of flounder, which is economical. High effect.
In addition, this marker may be able to determine flounder that is resistant to edovagerosis.

Using the genetic marker of marker locus Poli1506TUF on gene linkage group LG4, the genetic background of the susceptibility of Japanese flounder to edewellerosis is discriminated, and individuals susceptible to edewerosis are excluded and raised.
Examples of the present invention will be described in detail below.

The F ₁ (KP-CB) performs one-to-one mating was used to produce the F ₂ (KP-CBCB).
Using this F _2; KP-CBCB (n = 109, average body length of 13.2 cm) as a test fish, an infection test with E. tarda causing causative of Edovagerosis was performed. The test fish was immersed in a water bath with 8 × 10 ⁶ / ml of bacteria for 10 minutes to obtain phenotypic data for each individual. As a result, there were 17 live fish and 92 dead fish.
These test fish were subjected to PCR using PCR primers as follows, and the obtained PCR product was subjected to gel electrophoresis to confirm its genotype.

First, DNA extraction from all flounder was performed according to the following procedure.
A caudal fin was collected in a 1 cm square size, and 600 μL of a digestion solution containing 100 mM NaCl, 20 mM Tris-HCl (pH 8.0), 100 mM EDTA, 1.0% SDS, 100 μg / ml Proteinase K was added, and the mixture was added at 37 ° C. I left still overnight.
Further, after extraction with phenol / chloroform (1: 1) once, chromosomal DNA was precipitated by ethanol precipitation. The recovered DNA was washed with 70% ethanol, dried, and then dissolved in 50 μL of a TE solution (0.01 M Tris-HCl pH 7.4, 2.5 mM EDTA pH 8.0).

next,
F: 5′-TCT AGT GTT TGA GCA GAT AGG AAG GG-3 ′ (SEQ ID NO: 1)
R: 5′-CTC TTA GTG GCA CTC CAT CAC ACT G-3 ′ (SEQ ID NO: 2)
Synthesize a set of primers,
0.07 pmol F primer and [γ- ³³ P] ATP, 0.32 pmol R primer labeled with T4polynucleotide kinase, 0.2 mM each dNTP, 2.0
PCR was performed using Gene Amp PCR system 9600 thermal cycler (Perkin-Elmer) with 12 μl of a solution containing mM MgCl ₂ , 1% BSA, 0.25 U Ex Taq DNA polymerase (Takara Bio) and 50 ng template DNA. It was.

The PCR reaction composition (one sample) using this primer is shown in Table 1, and the RI primer solution composition (100 samples) of the R primer is shown in Table 2.

PCR conditions were 95 ° C., 2 minutes- (95 ° C., 30 seconds-62 ° C., 1 minute-72 ° C., 1 minute) × 35 cycles− (72 ° C., 3 minutes) × 1 cycle.
After the reaction, stir well with an equal amount of Loading dye (95% formamide, 10mD EDTA, 0.05% bromophenol blue and xylene cyanol), make the PCR product single-stranded by heat denaturation, and perform electrophoresis on 6% acrylamide gel. It was.
Thereafter, the gel dried for 1 hour is exposed to Imaging Plate (IP) for 3 to 12 hours, and the IP on which the photosensitive image of the radiation is stored is read by Bio-imaging Analyzer (BAS1000, Fuji Photo Films), and the image is displayed on a computer. Turned into.
The results are shown in FIGS.

As a result of electrophoresis, 41 individuals with both 148 bp and 151 bp as BB which is the marker type of the sensitive strain (KP-B) among the test fish, and the marker type of the disease resistant strain (KP-C) There were 12 individuals with only 142 bp as a certain CC. Further, as an F ₂ model analysis, the relationship between the presence of a band and the mortality rate was examined.
As a result, as is clear from Table 3, in the group having only 142 bp band, there are 4 surviving fish and 8 dead fish, whereas in the group having bands at 148 bp and 151 bp, 3 surviving fish are present. There were 38 tails and dead fish.
That is, the mortality rate in the group in which no band was confirmed in both 148 bp and 151 bp was 66.7%, and the mortality rate in the group in which the band was confirmed in 148 bp and 151 bp was 92.7%. The difference was obvious.

148bp and significance test of whether the Edwardsiella disease susceptibility 151bp band and 142bp bands, P value by chi ² square test analysis is 0.019, bands 148bp and 151bp PCR products using the primer There is a flounder that is prone to edegelosis.

In the above examples, 148 bp and 151 bp are shown as bands linked to the susceptibility to Edwardsia disease. However, the primers of SEQ ID NO: 1 and SEQ ID NO: 2 are designed as PCR primers including CA repeat sequences (microsatellite).
PCR primers containing microsatellite can locate one locus and detect many bands among individuals due to its polymorphism within the same species (DNAMakers: Protocols, Applications, and Overview, GUSTAVO CAETANO- ANOLLES PETER M.GRESSHOFF).
Therefore, the band size shown in this embodiment is not limited to this.
In addition, as a nucleic acid amplification reaction method, a LAMP method or the like can also be used. Furthermore, mass spectrometry, capillary electrophoresis, and the like can be used as an amplification product analysis method.

A gel electrophoretic profile of the present invention, the (a) is _{F 1 (KP-CB),} (b) dead individuals of _{F 2 (KP-CBCB),} (c) the F ₂ (KP-CBCB) Surviving individuals are shown. It is a schematic view of a gel electrophoresis of the present invention, (a) shows the _{F 1 (KP-CB),} (b) the dead individuals of _{F 2 (KP-CBCB),} (c) the F ₂ (KP-CBCB ) Surviving individuals, (d) indicates disease resistant strain (KP-C), sensitive strain (KP-B).

Claims (2)

  5′-TCT AGT GTT TGA GCA GAT AGG AAG GG-3 ′ (SEQ ID NO: 1), oligonucleotide portion consisting of at least 10 consecutive bases, and 5′-CTC TTA GTG GCA CTC CAT CAC ACT G -3 ′ (SEQ ID NO: 2), using the oligonucleotide portion consisting of at least 10 consecutive bases as a primer, a nucleic acid amplification reaction method using a flounder-derived nucleic acid as a template is performed, and the obtained amplification product is analyzed A method for discriminating the susceptibility of flounder to edewellerosis, characterized in that,   5′-TCT AGT GTT TGA GCA GAT AGG AAG GG-3 ′ (SEQ ID NO: 1), oligonucleotide portion consisting of at least 10 consecutive bases, and 5′-CTC TTA GTG GCA CTC CAT CAC ACT G -3 ′ (SEQ ID NO: 2), using the oligonucleotide part consisting of at least 10 consecutive bases as a primer, the polymerase chain reaction method is performed on the flounder genomic DNA, and the resulting product is subjected to gel electrophoresis. The method for determining susceptibility of flounder to Edwardla disease according to claim 1, wherein it is confirmed that there are bands at 148 bp and 151 bp.