CN115725722A - Detection reagent and detection kit for identifying bacterial gill disease tolerance of east Asian salmon - Google Patents
Detection reagent and detection kit for identifying bacterial gill disease tolerance of east Asian salmon Download PDFInfo
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Abstract
The invention belongs to the technical field of biological detection, and particularly relates to a detection reagent and a detection kit for identifying bacterial gill disease tolerance of eastern salmon. The invention discloses a detection reagent for identifying bacterial gill disease resistance of eastern Asian salmon, which screens out 104 SNP specific sites by performing transcriptome sequencing on head and kidney of each of susceptible and resistant 6 eastern Asian salmon, analyzes and verifies the SNP sites in groups by a gene sequencing method after multiple PCR and susceptible and resistant eastern Asian salmon samples, and screens out 6 SNP specific sites. The invention takes the 6 SNP loci as a kit detection item for screening bacterial gill disease resistance of east Asian salmon. The reagent and the kit have the advantages of short library construction period, good repeatability, simplicity and convenience in operation and the like, and can quickly screen out the Atlantic salmon with bacterial gill disease resistance on the premise of not damaging the Atlantic salmon, so that the Atlantic salmon breeding population with bacterial gill disease resistance is quickly established.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a detection reagent and a detection kit for identifying bacterial gill disease tolerance of eastern salmon.
Background
The Salmonidae fish is one of the 3 large farmed fishes in the world, and the farming yield is second to carp and tilapia. The breeding areas of China are mainly distributed in Heilongjiang, jilin, liaoning, gansu and other places, and the main varieties of the breeding are hucho salmon, rainbow trout, whitesalmon and Hongkong salmon. Atlantic Salmon (Salmon truta) also known as river Salmon and cat fish, belonging to Salmoniformes, salmonidae, salmonella, and is synonym with Oncorhynchus mykiss. The Atlantic salmon establishes relatively stable colonies in the Tibet Atlantic river, can grow and breed naturally, is an endemic variety, belongs to the cold water fishes, and is popular with consumers because of tender meat quality, no muscle prick, rich nutrition and delicious taste.
Bacterial Gill Disease (BGD) is a common disease in the farming of eastern salmon and, once it occurs, if a tangible measure is not taken quickly, it will cause serious damage to fishery development. However, there is currently no good method for detecting BGD resistance in Atlantic salmon.
Disclosure of Invention
The invention aims to provide a detection reagent and a detection kit for identifying bacterial gill disease resistance of east Asian salmon, and whether east Asian salmon has bacterial gill disease resistance can be quickly identified by a method of performing gene sequencing after multiple PCR by using the kit.
The invention provides a detection reagent for identifying bacterial gill disease tolerance of eastern salmon, which comprises specific primer pairs respectively designed aiming at 104 SNP sites;
the SNP locus is obtained by screening the individual eastern Asian salmon susceptible to bacterial gill disease and resistant to bacterial gill disease after transcriptome sequencing;
the transcriptome sequencing template is RNA of head and kidney tissues.
Preferably, the SNP sites are as follows:
the information of the SNP site is based on Salmo _ truntta.fSalTru 1.1 (http:// asia. Ensemblel. Org/Salmo _ truntta/Info/Index.
The invention also provides a detection kit for the bacterial gill disease resistance character of eastern salmon, which comprises the detection reagent.
The invention also provides a detection kit for the bacterial gill disease resistance character of eastern salmon, which comprises specific primer pairs respectively designed according to 6 SNP loci;
the 6 SNP loci comprise: chromosome 4 16842538-16842787, chromosome 12 77517014-77517244, chromosome 13 87581588-87581818, chromosome 18 18173575-18173832, chromosome 27 11029040-11028277 and chromosome 40 7014792-7015027.
Preferably, the nucleotide sequences of the specific primer pair designed for chromosome 4 16842538-16842787 are shown as SEQ ID NO.1 and SEQ ID NO.2, the nucleotide sequences of the specific primer pair designed for chromosome 12 77517014-77517244 are shown as SEQ ID NO.3 and SEQ ID NO.4, the nucleotide sequences of the specific primer pair designed for chromosome 13 87581588-87581818 are shown as SEQ ID NO.5 and SEQ ID NO.6, the nucleotide sequences of the specific primer pair designed for chromosome 18 18173575-1817373832 are shown as SEQ ID NO.7 and SEQ ID NO.8, the nucleotide sequences of the specific primer pair designed for chromosome 27 11029040-11028277 are shown as SEQ ID NO.9 and SEQ ID NO.10, and the nucleotide sequences of the specific primer pair designed for chromosome 40 7014792-7015027 are shown as SEQ ID NO.11 and SEQ ID NO. 12.
Preferably, the kit further comprises a reagent for gene sequencing after the multiplex PCR.
Preferably, the reagents for gene sequencing after multiplex PCR comprise independently packaged multiplex amplicon library building reagents and multiplex amplicon adapter reagents.
The invention also provides application of the reagent or the kit in creating germplasm of eastern salmon with bacterial gill disease resistance.
Has the advantages that: the invention provides a detection reagent for identifying bacterial gill disease resistance of eastern salmon, which comprises the steps of carrying out transcriptome sequencing on head and kidney of 6 eastern salmon susceptible and resistant to each bacterial gill disease, screening 104 SNP specific sites, designing primers, carrying out gene sequencing after multiple PCR (shown in figure 1) by a method, carrying out analysis and group verification on the 104 SNP sites by susceptible and resistant eastern salmon samples (30 tails of each group), finally screening 6 SNP specific sites, and preparing the SNP sites into a kit. For the eastern Asian salmon individual with unknown bacterial gill disease resistance, only about 0.5g of tissues (fat fins and the like) of the eastern Asian salmon are provided for DNA extraction, and the method of gene sequencing after multiplex PCR is carried out by a kit, so that whether the eastern Asian salmon has bacterial gill disease resistance can be rapidly identified. The kit is used for constructing a second-generation sequencing library through multiple PCR, is suitable for an Illumina platform, adopts two-round PCR reaction to construct the library, and has the advantages of short library construction period, good repeatability, simplicity and convenience in operation and the like. The detection kit provided by the invention is beneficial for researchers to quickly screen out the eastern salmon with bacterial gill disease resistance on the premise of not damaging the eastern salmon, so that a eastern salmon breeding population with bacterial gill disease resistance is quickly established, and the development of the farming of the eastern salmon is facilitated.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without inventive exercise.
FIG. 1 is a flow chart of a library building capture;
FIG. 2 shows a kit
Library Prep Kit(Module A Universal)。
Detailed Description
The invention provides a detection reagent for identifying bacterial gill disease resistance of eastern salmon, which comprises specific primer pairs respectively designed aiming at 104 SNP sites;
the SNP locus is obtained by screening after transcriptome sequencing of head and kidney of eastern Asian salmon individuals susceptible to bacterial gill disease and resistant to bacterial gill disease.
In the culture population of eastern salmon in eastern Asian county, 1 suspected pathogenic bacterium is separated and purified from gill-rot part of eastern salmon suffering from bacterial gill disease, and the bacterium is determined to be Aeromonas salmonicida (Aeromonas salmonida, ATCC 33658) through 16S rRNA gene sequencing comparison and identification.
The determination of whether or not the salmon has bacterial gill disease resistance by using the aeromonas salmonicida preferably includes infecting the salmon by using the aeromonas salmonicida based on an artificial infection experiment, wherein the salmon is determined to be an individual susceptible to bacterial gill disease if gill-rot or even dead salmon occurs, and the salmon having no obvious abnormality at gills and surviving is determined to be an individual having bacterial gill disease resistance.
In the present invention, the head and kidney of each 6-tailed east Asian salmon susceptible and resistant to the artificial infection experiment were subjected to transcriptome sequencing, and 104 SNP-specific sites were selected based on Salmo _ trutta.fSalTru1.1 (http:// asia. Ensembl. Org/Salmo _ trutta/Info/Index), as follows.
The invention also provides a detection kit for the bacterial gill disease resistance character of eastern salmon, which comprises the detection reagent.
The detection kit disclosed by the invention preferably detects the tolerance of the eastern salmon to the bacterial gill disease character by adopting a gene sequencing mode after multiple PCR, and the specific flow is shown in figure 1. According to the invention, based on the specific locus information of the 104 SNP loci, primers are designed, a gene sequencing method is carried out after multiple PCR, susceptible and resistant Atlantic salmon samples (30 tails of each group) obtained through an artificial infection experiment are supplemented, the 104 SNP loci are analyzed and subjected to group verification, finally 6 SNP specific loci are screened out, and the SNP loci can be used for screening the bacterial gill disease resistance of the Atlantic salmon, and are prepared into a commercialized kit.
The invention also provides a detection kit for the bacterial gill disease resistance character of the east Asian salmon, which comprises specific primer pairs respectively designed aiming at 6 SNP loci;
the 6 SNP sites comprise: chromosome 4 16842538-16842787, chromosome 12 77517014-77517244, chromosome 13 87581588-87581818, chromosome 18 18173575-18173832, chromosome 27 11029040-11028277 and chromosome 40 7014792-7015027.
TABLE 1 primer information of the detection kit
The detection kit of the invention preferably comprises three main parts which are independently packaged, and respectively comprise a reagent (a) for establishing a multi-amplicon library
Library Prep Kit), multiplex amplicon primer set(s) ((ii)
Primer Pool) and multiplex amplicon adapter assayAgent (A), (B)
Linker kit).
In the present invention, it is preferable that,
the Library Prep Kit (FIG. 2) is divided into two sub-modules, module A and Module B, wherein
The Library Prep Kit (Module A Universal) comprises the reagents shown in Table 2, and the storage temperature is recommended to be-20 ℃ +/-5 ℃, the storage condition of Enhancer Buffer M (2M betaine) is recommended to be 2-8 ℃, and the storage temperature can also be-20 ℃ +/-5 ℃; enhancer buffer NB 0.1mg/ml BSA.
TABLE 2
Library Prep Kit(Module A Universal)
The invention is as described
The Library Prep Kit (Module B) 960rxn can be packaged and divided into Module B1 and Module B2, wherein the Module B1 comprises IGT TM EM808 Polymerase mix and Enhancer Buffer M, module B2 contains YF Buffer B. The recommended storage conditions of Enhancer Buffer M and YF Buffer B are 2-8 ℃, and can also be stored at-20 ℃ +/-5 ℃.
TABLE 3
Library Prep Kit(Module B)
The invention
The composition of Primer Pool is shown in Table 4, where N in Table 4 is the mark of the branch pipe, according to the design of the branch pipe,
the Primer Pool comprises different combinations of T1, T1-T2, T1-T3 and the like. The total amount is the total amount of each tube-specific primer pool.
TABLE 4
Primer Pool
The invention is as described
The joint kit can be selected according to the experiment requirement
CDI Primer or
Any one of Dual-Indexed Primer; and in tables 5 and 6, N is divided into four combinations of 1-96, 97-192, 193-288, 289-384 according to the kit.
TABLE 5
CDI Primer
TABLE 6
Dual-Indexed Primer
The detection kit is suitable for
Custom Panel (A412 XV 1), illumina sequencing platform, specifically:
custom Panel (A412 XV1, for Illumina) can be used for second generation sequencing library construction by multiplex PCR, and is suitable for Illumina platform. The kit carries out primer design on a target region genome of interest based on a multi-factor algorithm, synthesizes effective specific primers, and further amplifies a target sequence, so that the sequencing data volume is greatly reduced, and only important regions are sequenced and analyzed; has huge application space in the aspects of clinical diagnosis and drug development, and also has wide application potential in related research in the field of animals and plants. The kit adopts two rounds of PCR reactions to construct the library, and has the advantages of short library construction period, good repeatability, simple and convenient operation and the like.
The invention also provides application of the reagent or the kit in creating east Asian salmon germplasm with bacterial gill disease resistance.
When the bacterial gill disease resistance is detected, the detection is carried out based on the 104 screened SNP loci, particularly the polymorphism information of the SNP loci, and part of the polymorphism information is shown in Table 7. In the invention, the primers of the 104 SNP sites pass through website (A), (B), (C), (D), (E) and (D)https://m.primer.igenetech.com/) Designing according to the instructions on the website, namely inputting the target area in BED format after logging in, and then selecting the species as Atlantic salmon, the number of tubes is SNPThe number of the primer sequences and other parameters are defaulted, and after submission, the primer sequences with the top rank are selected.
TABLE 7 differential SNP site information selected by Artosalmon head-kidney transcriptome sequencing (section)
In the present invention, when bacterial gill disease tolerance of created eastern salmon germplasm is detected, preferably, DNA extracted from eastern salmon tissue is used as a template, and the eastern salmon tissue is preferably about 0.5g of fat fin, and the like, a first round multiplex PCR reaction system is first constructed using the template, and the first round multiplex PCR reaction system is preferably 30 μ l in volume and comprises: enhancer buffer NB (1N) 3.5. Mu.l, enhancer buffer M2.5. Mu.l, primer pool 5. Mu.l, template 40ng, IGT-EM808 polymerase mix 10. Mu.l and the balance ddH 2 And O. The first round of multiplex PCR reaction is carried out by utilizing the constructed first round of multiplex PCR reaction system, and the first round of multiplex PCR reaction system preferably comprises a 105 ℃ hot cover (Heat lid); 3min at 95 ℃ for 30s; circulating at 98 ℃ for 20s and 60 ℃ for 4min, and circulating at 18 ℃; 5min at 72 ℃.
The invention carries out magnetic bead purification on the products of the first round of multiplex PCR reaction to obtain purified first round products, and then carries out second round of adaptor sequence PCR by utilizing the purified first round products, wherein the system of the second round of adaptor sequence PCR is counted by 30 mu l, and preferably comprises the following components: purify the first round product 13.5. Mu.l, enhancer buffer M2.5. Mu.l, CDI Primer (premixed adaptor Primer, concentration 5. Mu.M each) 2. Mu.l, IGT-EM808 polymerase mix 10. Mu.l and the remainder ddH 2 And (O). The second round adapter sequence PCR procedure of the present invention preferably comprises a 105 ℃ hot lid (Heat lid); 3min at 95 ℃ for 30s; circulation is carried out at the temperature of 98 ℃ for 20s, at the temperature of 58 ℃ for 1min, at the temperature of 72 ℃ for 30s and 9; 5min at 72 ℃. According to the invention, the second round of magnetic bead purification is carried out on the second round of adaptor sequence PCR product, and then the product is put on a computer to carry out library concentration and quality inspection. The joint primers are all generalThe sequence is obtained and downloaded from the illumina officer by a linker and synthesized.
IGT is preferred in the same embodiment of the two-round magnetic bead purification method of the present invention TM And purifying magnetic Beads or Agencour AMPure XP, storing the purified magnetic Beads at 4 ℃, taking out, performing vortex oscillation on the resuspended magnetic Beads, then placing the resuspended magnetic Beads at room temperature for 25-30 min to balance the magnetic Beads, and performing vortex oscillation on the resuspended magnetic Beads before use, so as to ensure that the magnetic Beads are uniform and have consistent concentration. The operation of magnetic bead purification according to the present invention preferably includes: 1) 80% ethanol was prepared in advance with absolute ethanol and Nuclear-Free Water, and left at room temperature for use. 2) Taking out purified magnetic beads from a refrigerator at 4 ℃ in advance, mixing uniformly, and placing at room temperature for balancing for 30min; the purified beads, which had equilibrated to room temperature, were vortexed and mixed until ready for use. 3) To the product to be purified, 0.9 volume of magnetic beads (27. Mu.L) was added, pipetted or vortexed and allowed to stand at room temperature for 5min. 4) And (5) performing instantaneous centrifugation, placing the PCR tube on a magnetic frame for 3min, and standing until the solution is clarified. 5) The supernatant was removed completely, the PCR tube was removed from the magnetic stand, 50. Mu.L of YF buffer B was added to the tube, pipetted and mixed well, and allowed to stand at room temperature for 5min. 6) And (5) performing instantaneous centrifugation, and placing the PCR tube on a DynaMag-96 Side magnetic frame for 3min. 7) The PCR tube was held on a magnetic holder, the supernatant was carefully removed, 180. Mu.L of 80% ethanol solution was added to the PCR tube, and the tube was allowed to stand for 30 seconds. 8) Keeping the PCR tube on the magnetic frame, removing the supernatant, adding 180 mu L of 80% ethanol solution into the PCR tube again, standing for 30s, and removing the supernatant. 9) Cover the tube cap, centrifuge instantaneously, centrifuge the residual ethanol to the bottom of the tube, place the PCR tube on a magnetic rack, carefully use a 10. Mu.L pipette to remove the residual ethanol at the bottom, and take care not to attract the beads. 10 Keeping the PCR tube on a magnetic frame, standing at room temperature for 3-5 min, and airing the magnetic beads to completely volatilize the residual ethanol. 11 Add 24. Mu.L of Nuclear-Free Water, remove the PCR tube from the magnetic stand, pipette or vortex well, and let stand at room temperature for 2min.12 ) centrifugation, place the PCR tube on a magnetic rack for 2min until the solution is clear. 13 Pipette 13.5. Mu.L of the supernatant, transfer to a new PCR tube, and mark the supernatant in the tube as a purified multiplex PCR product.
For further illustration of the present invention, the following detailed description of the detection reagent and detection kit for identifying bacterial gill disease resistance of eastern salmon provided in the present invention is provided in connection with the accompanying drawings and examples, which should not be construed as limiting the scope of the present invention.
Example 1
1 materials and methods
1.1 Experimental materials
All the salmon used in the experiment are from the breeding base of the institute of aquatic science and research of the academy of farming and animal husbandry in the autonomous region of Tibet, 24 healthy adult salmon (the physique is 360.00 +/-56.78 g, the full length is 30.17 +/-1.72 cm, and the body length is 27.33 +/-1.63 cm) are randomly selected to carry out an experiment for killing the artificial infection of the aeromonas salmonicida (the early experiment proves that the aeromonas salmonicida is the pathogenic bacteria of the bacterial gill disease of the salmon, the content is published on the journal of the aquatic science of Sunjiashuai, the week construction, wanliang and the like, and the separation and identification of pathogenic bacteria of the bacterial gill disease of the salmon [ J/OL ] aquatic science: 1-14 2022-09-30]. DOI: 10.16378/j.cnki.1003-1111.52 ]. Salmon showing bacterial gill disease and no symptoms were selected at 6 tails each to constitute a susceptible group and a resistant group, and head and kidney tissues of 2 groups of salmon were collected, respectively. After being collected, the sample is frozen in liquid nitrogen, and is transferred to a refrigerator at minus 80 ℃ for storage after 30 min.
1.2 Experimental methods
The experimental material is sent to Shanghai Senno biotechnology and science and technology Limited to perform transcriptome sequencing, and SNP loci related to bacterial gill disease resistance are screened from the obtained differential genes.
2 results
2.1 differential Gene results
Compared with the susceptible group and the resistant group, 1851 differential genes were co-screened, wherein 1026 genes were up-regulated and 825 genes were down-regulated.
2.2SNP site information
Statistical analysis is carried out on the SNP loci of each eastern Asian salmon, and comparison is carried out in 1851 difference genes, and finally 104 SNP loci with different genotypes between a susceptible group and a resistant group are obtained.
The 104 SNP loci screened by the above method (some are shown in Table 7) are selected by website: (https:// m.primer.igenetech.com/) Designing corresponding primers byThe multiplex PCR method comprises performing SNP identification and screening on 6 samples of each of the susceptible and resistant groups, and screening 6 SNP sites (Table 1), wherein the 6 sites have different genotypes between the susceptible group and the resistant group. Therefore, the 6 SNP loci can be preliminarily judged to be used as SNP loci for screening bacterial gill disease resistance of east Asian salmon.
Example 2
The 6 SNP loci selected in example 1 are used as detection bases, and the genotypes of the 6 SNP loci of 60 samples, 30 of which are susceptible and resistant respectively, are detected by a multiplex PCR method. The method comprises the following steps: 1.1 st round of multiplex PCR reaction
The process was carried out according to the instructions provided previously, with the reagent ratios as shown in the following table:
| Reagent | Volume(μl) |
| ddH 2 O | 9-x |
| Enhancer buffer NB(1N) | 3.5 |
| Enhancer buffer M | 2.5 |
| |
5 |
| sample(s) [1] | X |
| IGT-EM808polymerase mixture | 10 |
| Total | 30 |
[1] The initial amount is 40 ng/reaction tube; the concentration of DNA was the quantitative result of the Qubit (Thermo Fisher).
Wherein, enhancer Buffer NB (1N), enhancer Buffer M and IGT TM EM808 Polymerase mix is from kit
Library Prep Kit (Module A Universal). Primer Pool from kit
Primer Pool, which is a mixture of 6 selected SNP primers.
2. Round 1 magnetic bead purification
As per the instructions provided previously.
3. 2 nd round linker sequence PCR reaction
In accordance with the instructions provided previously, the reagent ratios are shown in the following table:
[1] the PCR product mix is a multiplex PCR product after the previous purification step.
[2] CDI Primer is premixed adaptor Primer with concentration of 5 μ M each
Wherein, IGT TM EM808 Polymerase mix and Enhancer Buffer M from kit
Library Prep Kit (Module A Universal). CDI Primer from kit
In the CDI Primer, since the linker sequence of only 6 SNP sites is used for verification, N may be selected from the combinations of 1 to 96.
4. Round 2 magnetic bead purification
As per the instructions provided previously.
5. Concentration measurement and quality control
As per the instructions provided previously.
6. Sequencing on machine
Obtaining PCR product sequence, analyzing SNP locus information, and carrying out reverse comparison.
The results show that: in30 susceptible group samples, 26 susceptible genotypes of 6 SNP loci are completely combined, and the accuracy rate is 86.67%; in30 resistant group samples, 27 resistant genotypes of 6 SNP loci are combined completely, and the accuracy rate is 90.00%. The remaining 7 tails did not completely match any of the genotype combinations and bacterial gill disease resistance was not judged by this method.
Although the present invention has been described in detail with reference to the above embodiments, it is only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and all of the embodiments are included in the scope of the present invention.
Claims (8)
1. The detection reagent is used for identifying that eastern salmon is resistant to bacterial gill disease, and is characterized by comprising specific primer pairs which are respectively designed aiming at 104 SNP sites;
the SNP locus is obtained by screening the individual eastern Asian salmon susceptible to bacterial gill disease and resistant to bacterial gill disease after transcriptome sequencing;
the template for transcriptome sequencing comprises RNA of the head kidney tissue.
2. The detection reagent according to claim 1, wherein the SNP sites are as follows:
the information of the SNP site is based on Salmo _ trutta. FSalTru1.1.
3. A test kit for detecting bacterial gill disease resistance of eastern salmon, which comprises the test reagent according to claim 1 or 2.
4. A detection kit for bacterial gill disease resistance of east Asian salmon is characterized by comprising specific primer pairs respectively designed aiming at 6 SNP sites;
the 6 SNP sites comprise: chromosome 4 16842538-16842787, chromosome 12 77517014-77517244, chromosome 13 87581588-87581818, chromosome 18 18173575-18173832, chromosome 27 11029040-11028277 and chromosome 40 7014792-7015027.
5. The detection kit according to claim 4, wherein the nucleotide sequences of the specific primer pair designed for chromosome 4 16842538-16842787 are shown as SEQ ID NO.1 and SEQ ID NO.2, the nucleotide sequences of the specific primer pair designed for chromosome 12 77517014-77517244 are shown as SEQ ID NO.3 and SEQ ID NO.4, the nucleotide sequences of the specific primer pair designed for chromosome 13 87581588-87581818 are shown as SEQ ID NO.5 and SEQ ID NO.6, the nucleotide sequences of the specific primer pair designed for chromosome 18 18173575-18173737373832 are shown as SEQ ID NO.7 and SEQ ID NO.8, the nucleotide sequences of the specific primer pair designed for chromosome 27 11029040-11028277 are shown as SEQ ID NO.9 and SEQ ID NO.10, and the nucleotide sequences of the specific primer pair designed for chromosome 40 7014792-7015027 are shown as SEQ ID NO.11 and SEQ ID NO. 12.
6. The detection kit according to claim 3 or 4, further comprising a reagent for gene sequencing after multiplex PCR.
7. The detection kit of claim 6, wherein the reagents for gene sequencing after multiplex PCR comprise separately packaged multiplex amplicon banking reagents and multiplex amplicon adapter reagents.
8. Use of a reagent according to claim 1 or 2 or a kit according to claim 3 or a kit according to any one of claims 4 to 7 for creating germplasm of eastern salmon that is resistant to bacterial gill disease.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117701729A (en) * | 2023-12-15 | 2024-03-15 | 华中农业大学 | SNP locus and locus combination related to bacterial gill rot disease resistance of grass carp and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117701729A (en) * | 2023-12-15 | 2024-03-15 | 华中农业大学 | SNP locus and locus combination related to bacterial gill rot disease resistance of grass carp and application thereof |
| CN117701729B (en) * | 2023-12-15 | 2024-05-03 | 华中农业大学 | SNP locus and locus combination related to bacterial gill rot disease resistance of grass carp and application thereof |
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