CN116516028A - SNP locus related to anti-nervous necrosis virus character of leopard gill-acanthus japonicus and application thereof - Google Patents
SNP locus related to anti-nervous necrosis virus character of leopard gill-acanthus japonicus and application thereof Download PDFInfo
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Abstract
The invention provides SNP loci related to anti-nervous necrosis virus traits of leopard gill-acanthus, and application thereof, wherein the SNP loci comprise 6SNP loci, and sequences of base fragments before and after each SNP locus are shown as SEQ ID NO. 1-SEQ ID NO. 6. The SNP molecular marker provided by the invention can be used for auxiliary breeding of the molecular marker of the leopard-gill-acanthus, quickens breeding of disease-resistant leopard-gill-acanthus varieties, and provides a reference basis for disease-resistant breeding work of the leopard-gill-acanthus.
Description
Technical Field
The invention belongs to the field of aquatic organism breeding, and particularly relates to SNP loci related to anti-nervous necrosis virus traits of leopard gill acanthus, and application thereof.
Background
Bao gill-acanthus japonicusPlectropomus leopardus) Belonging to the genus Perciformes, the order Perciformes, the subfamily Pericaceae, the genus Perciformes, commonly known as Dongxing plaque. The method is mainly distributed in tropical sea areas from Indian ocean to Pacific ocean, and is distributed in southwest coast and south sea areas, so that the method is rare in quantity of tropical coral reef fishes, high in nutritive value, delicious in meat quality and wide in market prospect. The Hainan of China has excellent natural conditions suitable for the growth of the leopard gill-like acanthus, the breeding industry of the Hainan has rapid development, and the breeding and artificial seedling raising are carried out at present, so that the supply of seedlings is not required.
In recent years, the virus nerve necrosis disease is found to be serious in the breeding process of the groupers, the virus nerve necrosis disease is caused by the infection of the nerve necrosis virus (nervous necrosis viral, NNV), and the biggest damage to the groupers is the red spot nerve necrosis virus genotype (Red spotted grouper NNV, RGNNV). The virus mainly affects the fries and young fishes in the production period of the grouper fries, so that the brains and retina of the fries are necrotic and cavitation, the fries die due to disease, the symptoms are anorexia, protruding eyeballs, unbalanced fish bodies, rolling sideways or irregular swimming, and the like during disease, the death rate of infection is over 90 percent, and the cultured fries can be all killed within one week when the death rate is most serious, so that the yield of the Dongxingby is seriously affected.
At present, no effective treatment method is available for the nervous necrosis virus, so that the cultivation of disease-resistant varieties is developed, and the improvement of the yield of the leopard gill-acanthus bass is an economic and effective mode. SNPs refer to variation of a single nucleotide in the genome, one of the most common forms of genetic variation. The research of SNP loci can reveal genetic differences among different individuals, and provides an important basis for the research of genetic characters. In the leopard gill-acanthus bass, the correlation between SNP loci and NNV resistance characteristics is researched, so that an important theoretical basis can be provided for disease-resistant breeding of the leopard gill-acanthus bass. Therefore, the research on the related SNP locus of the anti-NNV of the acanthus leopard bass has important significance for the cultivation and disease prevention and control of the acanthus leopard bass. At present, researchers have identified some SNP sites related to the anti-NNV of the groupers, but SNP sites related to the anti-nerve necrosis virus of the pyracantha leopard bass are not reported.
Disclosure of Invention
In view of the defects of the prior art, the invention provides SNP loci related to anti-nervous necrosis viruses of the leopard-gill-spiny bass and application thereof, and SNP molecular markers with breeding value are obtained by performing whole genome re-sequencing on young fishes of the anti-and susceptible leopard-gill-spiny bass and finding out key genes affecting disease resistance through whole genome association analysis. The method provided by the invention identifies SNP loci related to the anti-nervous necrosis virus of the leopard gill-acanthus, and excavates related candidate genes, so that technical support can be provided for breeding disease-resistant and high-quality groupers.
The technical scheme of the invention is as follows:
the invention aims to provide SNP loci related to anti-nervous necrosis viruses of the leopard gill acanthus bass, which comprises 6SNP loci, wherein the information of each locus is as follows:
the 1 st SNP locus is positioned at the 301 st position of the sequence shown in SEQ ID NO.1, wherein the base y at the 301 st position is c (cytosine) or t (thymine);
the SNP site 2 is positioned at the 300 rd position of the sequence shown in SEQ ID NO. 2, wherein the base r at the 300 rd position is a (adenine) or g (guanine);
the 3 rd SNP locus is positioned at the 300 rd position of the sequence shown in SEQ ID NO. 3, wherein the base y at the 300 rd position is c (cytosine) or t (thymine);
the 4 th SNP locus is positioned at the 301 st position of the sequence shown in SEQ ID NO. 4, wherein the base y at the 301 st position is c (cytosine) or t (thymine);
the 5 th SNP locus is positioned at the 301 st position of the sequence shown in SEQ ID NO. 5, wherein the base k at the 301 st position is t (thymine) or g (guanine);
the 6 th SNP site is located at the 301 st position of the sequence shown in SEQ ID NO.6, wherein the base s at the 301 st position is c (cytosine) or g (guanine).
Further description:
when the genotype of the 1 st SNP locus of the sample is TT, the genotype of the 2 nd SNP locus is AG, the genotype of the 3 rd SNP locus is TC, the genotype of the 4 th SNP locus is CC, the genotype of the 5 th SNP locus is GG or the genotype of the 6 th SNP locus is CG, the sample is an individual resisting the nerve necrosis virus;
when the genotype of the 1 st SNP locus of the sample is CT, the genotype of the 2 nd SNP locus is GG, the genotype of the 3 rd SNP locus is CC, the genotype of the 4 th SNP locus is TC, the genotype of the 5 th SNP locus is TG or the genotype of the 6 th SNP locus is GG, the sample is an individual susceptible to the nervous necrosis virus.
The second object of the present invention is to provide a primer combination for detecting SNP sites related to anti-nervous necrosis virus of the leopard gill acanthus, comprising:
the primer pair for detecting the 1 st SNP locus has an upstream primer sequence shown as SEQ ID NO.7 and a downstream primer sequence shown as SEQ ID NO. 8;
the primer pair for detecting the SNP locus of the 2 nd is shown in SEQ ID NO.9 as an upstream primer sequence and SEQ ID NO.10 as a downstream primer sequence;
the primer pair for detecting the 3 rd SNP locus has an upstream primer sequence shown as SEQ ID NO.11 and a downstream primer sequence shown as SEQ ID NO. 12;
the primer pair for detecting the 4 th SNP locus has an upstream primer sequence shown as SEQ ID NO.13 and a downstream primer sequence shown as SEQ ID NO. 14;
the primer pair for detecting the 5 th SNP locus has an upstream primer sequence shown as SEQ ID NO.15 and a downstream primer sequence shown as SEQ ID NO. 16;
the primer pair for detecting the 6 th SNP locus has an upstream primer sequence shown as SEQ ID NO.17 and a downstream primer sequence shown as SEQ ID NO. 18.
The invention further aims to provide an application of the SNP locus related to the anti-nervous necrosis virus character of the leopard-gill-spine bass or the primer combination in breeding of disease-resistant varieties of the leopard-gill-spine bass.
Further, the disease-resistant variety is a variety resistant to nervous necrosis virus.
Compared with the prior art, the invention has the beneficial effects that:
the SNP molecular markers obviously related to the anti-nerve necrosis virus of the Larix parviflorus are screened out, 7 disease-resistant related genes are positioned, the related SNP molecular markers and genes can be used for auxiliary breeding of Larix parviflorus molecular markers, breeding of disease-resistant Larix parviflorus varieties is quickened, and a reference basis is provided for disease-resistant breeding work of Larix parviflorus or other cultured fishes.
Drawings
FIG. 1 is a principal component analysis chart of disease resistant and susceptible groups.
FIG. 2 is a Manhattan plot of genome-wide association analysis of disease resistant and susceptible groups.
Detailed Description
The present invention will be further described with reference to specific examples and drawings for a better understanding of the technical contents of the present invention to those skilled in the art.
Example 1 Whole genome association analysis of anti-Neuronecrosis Virus of Tripterygium parvum
5 months 2022, the eastern city of Hainan province shows that juvenile fish of the Perch, which is bred by the limited company of the morning sea water factory in Hainan province, have viral nervous necrosis symptoms, such as: anorexia, eyeball herniation, loss of balance of fish body, rolling on the body or irregular swimming, etc. Selecting 100 dead juvenile fish of the leopard-gill-acanthus bass after virus infection as a susceptible group, selecting 100 juvenile fish of the susceptible group and 100 juvenile fish of the resistant group as a disease resistant group, sending 200 juvenile fish samples to Shenzhen Huada company for sequencing, carrying out genome re-sequencing and GWAS analysis on the susceptible group and the disease resistant group, and carrying out sequencing by a combined probe anchored polymerization technology (cPAS) with a sequencing depth of 5×.
1.1 sequencing data quality control
And (3) performing quality control on raw data (raw data) obtained by sequencing, filtering and performing quality control by using SOAPnuke software, removing reads with low quality, more N bases and joint pollution, using the filtered high-quality reads for subsequent analysis, and performing relevant data statistics on the obtained clean data (effective data), wherein the statistics comprise sequencing data quantity, Q20 proportion, Q30 proportion, GC content and the like.
1.2 genome alignment
And (3) comparing the clean ready with the genome of the leopard gill-acanthus by using BWA software (BWA mem-M-t 40) to obtain a sam file. Converting the sam files into the bam files by using a Samtools tool, sequencing the bam files and creating an index; the Markdaulicates tool in Picard was used to delete PCR repeat reads that are typically generated during library construction.
1.3 mutation detection and quality control
SNP and InDel detection is carried out on the data after the duplication removal by using the mp call parameter in Bcftools; extracting SNPs from Vcftools to vcf files, and further performing strict quality control on SNPs obtained through preliminary detection to ensure accuracy of mutation information detection. Quality control is carried out on SNP loci by using Plink (v1.90b6.12) software, SNP loci meeting the standard are screened, and the quality control standard is as follows: the minimum allele frequency >0.05, followed by hardy-weinberg (hadi-winberg) equilibrium filtration with a site deletion rate <20%, obtaining 1550475 sites for subsequent GWAS analysis, PCA in PLINK, and visualization of PCA plots using ggplot2 package (R language package), as in fig. 1, with no stratification, even distribution of resistance and susceptible individuals.
The genome-wide association analysis was performed using Linear Mixed Model (LMM) functions in the GEMMA v0.98.1 software to combine SNP genotype and phenotype data. A manhattan diagram created using the ggplot2 package is shown in fig. 2.
1.4 Screening of genes
The threshold was adjusted according to manhattan diagram, set to-log 10 (P-value) =4 as a statistically significant threshold, and sites where the P value reached the threshold were considered to have significant correlation.
The results show that 19 SNP loci in 1550475 SNPs meet the requirements, and 7 candidate genes and SNP information possibly related to anti-nervous necrosis virus are primarily identified according to physical information and gene annotation by Blast analysis and comparison. The information of these 7 SNPs, including SNP physical location, chromosomal location, recent gene and gene location, etc., are shown in Table 1. They are related to the property of the anti-nerve necrosis virus of the Larix parviflorus, and can be used for breeding the disease resistance property of the Larix parviflorus.
TABLE 1 significant SNP information mapping to genes
1.5 Results
Whole genome association analysis mapped to 7 genes associated with anti-nerve necrosis virus: vipr2, herc2, pip5k1c, sik2b, anp, mybpc3, arhgap9. The vipr2 is a pseudogene, the here 2 and pip5k1c genes participate in regulating immune cells, the important role is probably played in the response of juvenile fish of the leopard gill-red bass to the infection of the nervous necrosis virus, the nervous necrosis virus infects fish bodies through a central nervous system, target organs are fish brains and eyes, brain tissues have the characteristics of high oxygen consumption, moderate oxidation resistance, defence capacity and the like, so that the brain tissues are easily affected by the oxidation stress, the brain injury is always considered to be related to the oxidation stress, the oxidative stress of fish bodies after virus invasion and tissue hypoxia can cause ischemic neuron injury of the fish brains, the survival of the neurons is affected, and the sik2b plays an important role in regulating the survival of the neurons and can be used as a potential target for improving the survival rate of the neurons.
Example 2 disease resistance selection Using SNPs of the related genes of the anti-nerve necrosis Virus of the Larix parvus
The genome-wide association analysis is carried out, and 6SNP loci with obvious association disease resistance genes are screened out, wherein the SNP loci are shown in a table 2, are associated with the disease resistance characters of the leopard gill acanthus bass, can be used for breeding the disease resistance characters of the leopard gill acanthus bass, and are analyzed, and the results are shown in a table 3.
TABLE 2 information on SNPs associated with genes related to anti-nerve necrosis virus by Perch
Table 36 genotype statistical analysis of SNP loci in anti-susceptibility and susceptible groups
As can be seen from table 3, the genotypes of the 6 SNPs sites were significantly different in both the anti-susceptibility group and the susceptibility group (P < 0.05), and the genotypes significantly associated with the disease resistance trait in the 6 SNPs sites were: chr3_33806863: TT, chr12-17717695-AG, chr5-17993736-TC, chr7-36063950: CC, chr1_ 16193961:GG, chr2_ 24149020:CG. Genotypes significantly associated with susceptibility are: chr3_ 33806863:CT, chr12_ 17717695:GG, chr5_ 17993736:CC, chr7_36063950: TC, chr1_ 16193961:TG, chr2_ 24149020:GG. Therefore, the SNPs mark can be used for breeding the anti-nervous necrosis disease character of the nervous necrosis virus.
Example 3A method for detecting resistance to nervous necrosis Virus of Larix parviflorus
Based on the 6 single nucleotide polymorphism sites positioned in the embodiment 2 of the invention, corresponding primers are developed and used for molecular marker assisted selective breeding of the disease-resistant leopard-gill-spine bass so as to obtain the leopard-gill-spine bass variety resistant to the nerve necrosis virus. Primer information is shown in Table 4.
TABLE 4 primer information
And (3) the young fishes of the sea water limited company in the morning of Hainan province of 2023 show the symptom of nervous necrosis, 100 diseased and dead young fishes are collected, 100 disease-resistant and survival young fishes are collected, and the disease resistance of the to-be-detected leopard-gill-spine bass to the nervous necrosis virus is determined by detecting 6SNP loci of the sample of the to-be-detected leopard-gill-spine bass in example 2.
The method comprises the following steps:
(1) Extracting genome DNA of the to-be-detected leopard gill acanthus bass;
(2) Respectively carrying out PCR amplification by taking the DNA obtained in the step (1) as a template and taking nucleotide sequences shown in SEQ ID NO. 7-8, 9-10, 11-12, 13-14, 15-16 and 17-18 as primers to obtain PCR amplification products;
(3) Sequencing the PCR amplification product obtained in the step (2), and determining genotypes of 6SNP loci of the to-be-detected leopard gill acanthus;
(4) Determining whether the to-be-detected leopard gill acanthus bass is resistant to the nervous necrosis virus according to the genotypes of the 6SNP loci determined in the step (3), wherein the survival rate and death rate statistical results are shown in table 4:
statistical analysis of disease-resistant genotypes of 5 6SNP loci on diseased Larix parviflorus
The survival probability after infection with the nervous necrosis virus is higher than the death probability when the genotype of the sample containing the SNP site chr3_33806863 is TT, when the genotype of the sample containing the SNP site chr12_17717695 is AG, when the genotype of the sample containing the SNP site chr5_17993736 is TC, when the genotype of the sample containing the SNP site chr7_36063950 is CC, when the genotype of the sample containing the SNP site chr1_16193961 is GG, and when the genotype of the sample containing the SNP site chr2_24149020 is CG.
The above embodiments are only examples of the present invention, and the present invention is not limited thereto, but any modifications, equivalents, improvements, etc. made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (4)
1. SNP loci related to the anti-nervous necrosis virus trait of the leopard gill-acanthus japonicus comprise 6SNP loci and are characterized in that:
the 1 st SNP locus is positioned at the 301 st position of the sequence shown in SEQ ID NO.1, wherein the base y at the 301 st position is c or t;
the SNP site 2 is positioned at the 300 rd position of the sequence shown in SEQ ID NO. 2, wherein the base r at the 300 rd position is a or g;
the 3 rd SNP locus is positioned at the 300 rd position of the sequence shown in SEQ ID NO. 3, wherein the base y at the 300 rd position is c or t;
the 4 th SNP locus is positioned at the 301 st position of the sequence shown in SEQ ID NO. 4, wherein the base y at the 301 st position is c or t;
the 5 th SNP locus is positioned at the 301 st position of the sequence shown in SEQ ID NO. 5, wherein the base k at the 301 st position is t or g;
the 6 th SNP locus is positioned at the 301 st position of the sequence shown in SEQ ID NO.6, wherein the base s at the 301 st position is c or g.
2. The SNP site associated with the anti-nervous necrosis virus trait of leopard gill-like acanthus according to claim 1, wherein:
when the genotype of the 1 st SNP locus of the sample is TT, the genotype of the 2 nd SNP locus is AG, the genotype of the 3 rd SNP locus is TC, the genotype of the 4 th SNP locus is CC, the genotype of the 5 th SNP locus is GG or the genotype of the 6 th SNP locus is CG, the sample is an individual resisting the nerve necrosis virus;
when the genotype of the 1 st SNP locus of the sample is CT, the genotype of the 2 nd SNP locus is GG, the genotype of the 3 rd SNP locus is CC, the genotype of the 4 th SNP locus is TC, the genotype of the 5 th SNP locus is TG or the genotype of the 6 th SNP locus is GG, the sample is an individual susceptible to the nervous necrosis virus.
3. The primer set for detecting the SNP site according to claim 1, comprising:
the primer pair for detecting the 1 st SNP locus has an upstream primer sequence shown as SEQ ID NO.7 and a downstream primer sequence shown as SEQ ID NO. 8;
the primer pair for detecting the SNP locus of the 2 nd is shown in SEQ ID NO.9 as an upstream primer sequence and SEQ ID NO.10 as a downstream primer sequence;
the primer pair for detecting the 3 rd SNP locus has an upstream primer sequence shown as SEQ ID NO.11 and a downstream primer sequence shown as SEQ ID NO. 12;
the primer pair for detecting the 4 th SNP locus has an upstream primer sequence shown as SEQ ID NO.13 and a downstream primer sequence shown as SEQ ID NO. 14;
the primer pair for detecting the 5 th SNP locus has an upstream primer sequence shown as SEQ ID NO.15 and a downstream primer sequence shown as SEQ ID NO. 16;
the primer pair for detecting the 6 th SNP locus has an upstream primer sequence shown as SEQ ID NO.17 and a downstream primer sequence shown as SEQ ID NO. 18.
4. Use of the SNP locus as set forth in any one of claims 1 to 2 or the primer combination as set forth in claim 3 in breeding of disease-resistant varieties of leopard gill-acanthus, which are varieties resistant to nervous necrosis virus.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116694785A (en) * | 2023-08-02 | 2023-09-05 | 中国海洋大学三亚海洋研究院 | Growth-related SNP molecular marker of leopard gill acanthus and application thereof |
| 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 |
Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103004668A (en) * | 2013-01-04 | 2013-04-03 | 莱州明波水产有限公司 | Plectropomus leopardus fry north industrialized high-density circulation water rearing method |
| CN103388027A (en) * | 2013-08-02 | 2013-11-13 | 厦门大学 | Identification method of single nucleotide polymorphism of plectropomus leopardus in different groups |
| CN103589802A (en) * | 2013-11-20 | 2014-02-19 | 陈双雅 | Primer pair for identifying plectropomus leopardus, probe, kit and detection method |
| CN104357547A (en) * | 2014-09-17 | 2015-02-18 | 中山大学 | Construction method of Plectropomus microsatellite DNA molecular markers |
| CN112501312A (en) * | 2020-12-21 | 2021-03-16 | 华南农业大学 | Reagent for detecting SNP molecular marker combination related to anti-nervous necrosis virus character of grouper |
| CN113166769A (en) * | 2018-11-01 | 2021-07-23 | 蓝色等位基因有限责任公司 | Methods for altering gene expression of genetic disorders |
| CN114015789A (en) * | 2021-12-06 | 2022-02-08 | 中国水产科学研究院黄海水产研究所 | Genome selection method for cultivating disease-resistant improved Dongxiang spots |
| CN114410746A (en) * | 2022-03-29 | 2022-04-29 | 中国海洋大学三亚海洋研究院 | Dongxiang spot molecule source-tracing selection breeding method and application thereof |
| CN114736969A (en) * | 2022-02-28 | 2022-07-12 | 华南农业大学 | Method for selecting anti-nervous necrosis virus individual of epinephelus lanceolatus |
| CN114891894A (en) * | 2022-04-20 | 2022-08-12 | 广东海洋大学 | SSR molecular marker primer group for identifying Guangxi \/28064;, Zhongdao leopard gill and acantho salmoides protospecies and application thereof |
| CN115896297A (en) * | 2022-05-23 | 2023-04-04 | 集美大学 | SNP marker for anti-nervous necrosis disease virus breeding of Epinephelus akaara and application thereof |
| CN116287465A (en) * | 2023-03-16 | 2023-06-23 | 海南大学 | Real-time quantitative PCR (polymerase chain reaction) detection primer and detection kit for garrupa iridovirus |
-
2023
- 2023-06-27 CN CN202310760826.7A patent/CN116516028B/en active Active
Patent Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103004668A (en) * | 2013-01-04 | 2013-04-03 | 莱州明波水产有限公司 | Plectropomus leopardus fry north industrialized high-density circulation water rearing method |
| CN103388027A (en) * | 2013-08-02 | 2013-11-13 | 厦门大学 | Identification method of single nucleotide polymorphism of plectropomus leopardus in different groups |
| CN103589802A (en) * | 2013-11-20 | 2014-02-19 | 陈双雅 | Primer pair for identifying plectropomus leopardus, probe, kit and detection method |
| CN104357547A (en) * | 2014-09-17 | 2015-02-18 | 中山大学 | Construction method of Plectropomus microsatellite DNA molecular markers |
| CN113166769A (en) * | 2018-11-01 | 2021-07-23 | 蓝色等位基因有限责任公司 | Methods for altering gene expression of genetic disorders |
| CN112501312A (en) * | 2020-12-21 | 2021-03-16 | 华南农业大学 | Reagent for detecting SNP molecular marker combination related to anti-nervous necrosis virus character of grouper |
| CN114015789A (en) * | 2021-12-06 | 2022-02-08 | 中国水产科学研究院黄海水产研究所 | Genome selection method for cultivating disease-resistant improved Dongxiang spots |
| CN114736969A (en) * | 2022-02-28 | 2022-07-12 | 华南农业大学 | Method for selecting anti-nervous necrosis virus individual of epinephelus lanceolatus |
| CN114410746A (en) * | 2022-03-29 | 2022-04-29 | 中国海洋大学三亚海洋研究院 | Dongxiang spot molecule source-tracing selection breeding method and application thereof |
| CN114891894A (en) * | 2022-04-20 | 2022-08-12 | 广东海洋大学 | SSR molecular marker primer group for identifying Guangxi \/28064;, Zhongdao leopard gill and acantho salmoides protospecies and application thereof |
| CN115896297A (en) * | 2022-05-23 | 2023-04-04 | 集美大学 | SNP marker for anti-nervous necrosis disease virus breeding of Epinephelus akaara and application thereof |
| CN116287465A (en) * | 2023-03-16 | 2023-06-23 | 海南大学 | Real-time quantitative PCR (polymerase chain reaction) detection primer and detection kit for garrupa iridovirus |
Non-Patent Citations (2)
| Title |
|---|
| XIN WEN 等: "Genome-wide association study of red skin color in leopard coral grouper (Plectropomus leopardus) based on genome resequencing", 《AQUACULTURE》, vol. 563, no. 2, pages 1 - 11 * |
| 周可欣 等: "豹纹鳃棘鲈体色变异的色素细胞差异分析", 《水生生物学报》, vol. 45, no. 5, pages 1164 - 1170 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116694785A (en) * | 2023-08-02 | 2023-09-05 | 中国海洋大学三亚海洋研究院 | Growth-related SNP molecular marker of leopard gill acanthus and application thereof |
| CN116694785B (en) * | 2023-08-02 | 2023-12-19 | 中国海洋大学三亚海洋研究院 | Growth-related SNP molecular marker of leopard gill acanthus and application thereof |
| 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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