CN109182545B - SNP marker associated with vibrio harveyi disease of large yellow croaker, primer and application thereof - Google Patents
SNP marker associated with vibrio harveyi disease of large yellow croaker, primer and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of molecular identification of fishes, in particular to an SNP marker associated with vibrio harveyi disease of large yellow croaker, a primer and application thereof, wherein the nucleotide sequence of the SNP marker is shown as SEQ ID NO.1, and the 342 th base position G or T of the sequence from the 5' end is provided. The invention overcomes the problem that the prior art has no SNP marker which can be used for breeding the disease-resistant related traits of the large yellow croaker, and the probability that the large yellow croaker with the genotype GT infects Vibrio harveyi at the SNP locus is obviously higher than that of a homozygous GG genotype individual. Therefore, whether the large yellow croaker is susceptible to Vibrio harveyi disease can be effectively determined by detecting the SNP. Elimination of GT genotype individuals in parent breeding is beneficial to improving the capability of offspring in resisting vibrio harveyi infection, and the marker of the invention is utilized for auxiliary breeding, thereby accelerating the culture process of disease-resistant varieties of large yellow croakers.
Description
Technical Field
The invention relates to the technical field of molecular identification of fishes, in particular to an SNP marker associated with vibrio harveyi disease of large yellow croaker, a primer and application thereof.
Background
Large yellow croaker (Larimichthys crocea) is one of the important offshore economic fishes in China. With the rapid expansion of the artificial breeding scale and the continuous improvement of the intensification degree of the large yellow croaker, the breeding diseases of the large yellow croaker caused by bacteria, parasites, viruses and the like are continuously appeared and become more serious. The vibriosis is long in onset time, high in incidence and mortality rate and becomes an important restriction factor for large yellow croaker culture industrialization, and the vibriosis harveyi caused by Vibrio harveyi is one of the most common infectious Vibrio diseases for culturing large yellow croakers. In order to control vibrio harveyi diseases, culturists often use a large amount of drugs such as antibiotics. Not only causes food safety problems of aquatic product drug residue and the like, but also causes a series of problems of increase of drug resistance of pathogenic microorganisms, water pollution, deterioration of breeding environment and the like. Therefore, screening molecular markers associated with disease resistance traits, and breeding the disease-resistant excellent variety of the large yellow croaker by marker-assisted breeding becomes one of effective ways for controlling disease occurrence.
Molecular Marker Assisted Selection (MAS) is a novel molecular breeding technical means for identifying genotypes of different individuals by using molecular markers closely linked with target genes so as to perform assisted selective breeding. The molecular marker assisted breeding can effectively combine genotype and phenotype identification, and avoids blindness and unpredictability of seed selection, so that the accuracy of selection and the breeding efficiency are remarkably improved, the breeding process is accelerated, and the breeding period is shortened.
The SNP marker is the most efficient marker after a microsatellite, has the advantages of high polymorphism, rich content, stable heredity, simple analysis and the like in the whole genome because of being a double-allelic marker, and is widely applied to selective breeding of various animals and a few fishes. However, SNP related to disease resistance of the large yellow croaker is not reported, and SNP markers which can be effectively used for breeding of disease resistance related traits of the large yellow croaker need to be excavated.
Disclosure of Invention
The invention aims to overcome the problem that an SNP marker which can be used for breeding disease-resistant related traits of large yellow croakers does not exist in the prior art, and provides an SNP marker related to vibrio harveyi of large yellow croakers, a primer and an application thereof, wherein the SNP marker can be effectively used for breeding disease-resistant traits of large yellow croakers and can be used for rapidly detecting the disease resistance of the large yellow croakers to the vibrio harveyi.
In order to achieve the purpose, the invention is realized by the following technical scheme:
an SNP marker related to Vibrio harveyi disease of large yellow croaker, the nucleotide sequence of the SNP marker is shown as SEQ ID NO.1, and the 342 th base position G or T of the sequence from the 5' end.
A primer pair for detecting the SNP marker, wherein the nucleotide sequence of the primer pair of the SNP marker is shown as SEQ ID NO. 2-3.
A kit for detecting the SNP marker, which comprises the primer pair.
The method for detecting the susceptibility of the large yellow croaker to the vibrio harveyi disease is characterized in that the large yellow croaker to be detected is subjected to the detection of the SNP marker, and whether the large yellow croaker to be detected is susceptible to the vibrio harveyi disease is determined.
Preferably, the detection method comprises the following steps:
(1) extracting the genome DNA of the large yellow croaker to be detected;
(2) carrying out PCR amplification on the DNA obtained in the step (1) by using a primer pair for detecting the SNP marker to obtain a PCR amplification product;
(3) sequencing the PCR amplification product obtained in the step (2), and determining the genotype of the SNP marker of the large yellow croaker to be detected according to the sequencing result;
(4) and (4) determining whether the large yellow croaker to be detected is susceptible to vibrio harveyi according to the genotype of the SNP marker determined in the step (3).
Preferably, the GT-individual genotype of the SNP marker is more susceptible to Vibrio harveyi disease in large yellow croaker. Namely, the probability that the large yellow croaker with the GT genotype of the SNP locus is infected with the Vibrio harveyi disease is obviously higher than that of other genotype individuals.
In the detection method for detecting the SNP associated with the vibrio harveyi disease of the large yellow croaker, the genome extraction is not particularly limited, the traditional phenol chloroform extraction method can be adopted, the kit extraction method can also be adopted, the specific embodiment of the invention adopts the kit extraction method, the kit extraction method is simple and convenient to operate and rapid, and the extracted DNA has high quality. In addition, the individual genotype detection method of the large yellow croaker to be detected is not particularly limited, and the techniques such as flight time mass spectrum, Taqman probe, single-strand conformation polymorphism polymerase chain reaction, direct sequencing and the like can be used for detecting SNP. Among them, direct sequencing is the most accurate method and is also the gold standard for SNP detection. Generally, the sequencing peak of a homozygous SNP site is a single peak type, while the sequencing peak of a heterozygous SNP site is a nested peak, and thus it is easily distinguished. The detection rate of SNP detection by a direct sequencing method is close to 100 percent. Therefore, the present invention employs a direct sequencing method to detect SNP markers.
The invention has the beneficial effects that: according to research, the probability that the large yellow croaker with the GT genotype is infected with the Vibrio harveyi disease at the SNP locus is obviously higher than that of a homozygous GG type individual. Therefore, whether the large yellow croaker is susceptible to vibrio harveyi can be effectively determined by detecting the SNP. Specifically, when the locus genotype is heterozygous GT, the large yellow croaker is easy to suffer from Vibrio harveyi, and the fact that the large yellow croaker is easy to suffer from Vibrio harveyi after the G mutation of the locus is T is shown. Therefore, the SNP marker is closely related to the Vibrio harveyi disease of the large yellow croaker, elimination of GT genotype individuals in parent breeding is beneficial to improving the capability of offspring of resisting the Vibrio harveyi infection, and the marker is beneficial to auxiliary breeding, thereby accelerating the culture process of disease-resistant fine varieties of the large yellow croaker.
The primer pair which is beneficial to the SNP locus can effectively detect the genotype of the large yellow croaker to be detected, and the result can judge whether the large yellow croaker to be detected is susceptible to vibrio harveyi on one hand and provide reference for parent selection on the other hand. Therefore, the SNP site primer can be used for detecting the genotype of the large yellow croaker, judging whether the individual is susceptible to vibrio harveyi or not, and can be effectively used for the molecular marker-assisted selective breeding of the large yellow croaker and accelerating the breeding process of disease-resistant excellent varieties of the large yellow croaker.
The invention also provides a kit for detecting the SNP marker, which can effectively detect the resistance of the large yellow croaker to the infection of the Vibrio harveyi and is used for the molecular marker-assisted breeding of the large yellow croaker.
Therefore, the invention has the following beneficial effects:
(1) can effectively determine whether the vibrio harveyi disease is susceptible to the vibrio harveyi disease;
(2) the capability of resisting the infection of the Vibrio harveyi of the offspring is improved;
(3) accelerating the culture process of disease-resistant varieties of the large yellow croakers.
Drawings
FIG. 1 is a PCR amplification electrophoretogram of 10 SNPs.
FIG. 2 is a peak diagram of SNP site sequencing.
Detailed Description
Hereinafter, embodiments of the present invention will be described in detail. The following examples are only for illustrating the technical solutions of the present invention more clearly, and therefore are only examples, and the protection scope of the present invention is not limited thereby.
1.1 Large yellow croaker population sample Source
Selecting large yellow croaker to be tested from 3-month-old large yellow croaker artificially bred in the same batch at aquatic research institute in Zhoushan city, randomly selecting 200 healthy large yellow croakers with weight of about 50 g/tail, and injecting 1.0 × 10 by intraperitoneal injection to the large yellow croaker70.2ml of vibrio harveyi bacterial liquid with cfu/ml concentration, wherein the individuals who die within 10 days and have typical vibrio harveyi infection symptoms are susceptible individuals, and the individuals without infection symptoms are disease-resistant individuals. 80 individuals of the susceptible group and the disease-resistant group are obtained, and the muscles are cut and preserved in absolute ethyl alcohol for genome DNA extraction.
1.2 Large yellow croaker genome DNA extraction to be tested
The genome DNA is extracted by a phenol-chloroform extraction method, and the specific method comprises the following steps: taking about 30mg of muscle tissue, adding 200ul of digestive juice (1L of digestive juice contains 5ml of 2mol/L NaCl, 5ml of 2mol/L Tris-Cl, 100ml of 5% SDS and 100ml of 1mol/L EDTA, pH is 8.0), 20ul of 10mg/ml proteinase K, and digesting for 1-3 hours in a water bath kettle at 56 ℃; adding 500ul (pH 8.0) of balance phenol, fully reversing and mixing uniformly, centrifuging at 12000g for 10min, and carefully sucking the supernatant; adding 500ul phenol-chloroform-isoamyl alcohol mixture (phenol: chloroform: isoamyl alcohol volume ratio is 25:24:1), fully reversing and mixing for 10min, centrifuging at 12000g for 10min, and carefully sucking the supernatant; adding 500ul of chloroform-isoamyl alcohol mixture (the volume ratio of chloroform to isoamyl alcohol is 24:1) and washing once; adding 500ul of anhydrous ethanol, centrifuging at 12000g for 5min, carefully pouring off ethanol, and keeping precipitate; adding 200ul 75% ethanol, slowly mixing, centrifuging at 12000g for 5min, and removing ethanol; washing with 75% ethanol repeatedly; air drying for 15min, adding 100ul deionized water for dissolving, detecting DNA extraction quality and concentration by agarose gel electrophoresis and ultraviolet spectrophotometer, and storing at-20 deg.C for use.
1.3 sequencing and SNP marker development
Randomly selecting 10 large yellow croakers of the susceptible group and the disease-resistant group, respectively carrying out PCR amplification (an electrophoresis chart is shown in figure 1) and direct sequencing (a sequencing peak chart is shown in figure 2) on immune related genes of each large yellow croaker, and carrying out comparison analysis and SNPs screening on obtained sequences of the 10 susceptible groups and the 10 disease-resistant groups to obtain 1 SNP locus related to Vibrio harveyi, wherein the SNP locus is positioned at the 342bp position from the 5' end of a nucleotide sequence (full length 545bp) of SEQ ID NO.1, the base of the locus is represented by K, and K represents G or T. The nucleotide sequence shown in SEQ ID NO.1 is derived from a large yellow croaker functional gene sequence obtained by previous research of the inventor.
1.4PCR amplification and detection are carried out by taking large yellow croaker genome DNA as a template and adopting primers shown in SEQ ID NO. 2-3.
The PCR reaction conditions are as follows: 5 minutes at 95 ℃; 30 seconds at 95 ℃, 30 seconds at 56 ℃, 20 seconds at 72 ℃ for extension, 32 cycles; extension at 72 ℃ for 10 min. The reaction system was calculated at 50. mu.L: mu.L of 0.5. mu.M primer, 25. mu.L of Taq polymerase premix (TaKaRa), 1. mu.L of 10ng genomic DNA, and 22. mu.L of sterile double distilled water. And (3) directly sequencing the PCR product by using an ABI 3730DNA sequence sequencer, and analyzing the site information of the SNP according to a sequencing peak map.
1.5 Association analysis of SNP loci and Vibrio harveyi disease
The genotype of the SNP site and the correlation between the allele and the disease resistance are analyzed by using multivariate analysis of variance and independent sample T test in a general linear model of SPSS 19.0, and the analysis results of the difference between the allele and the genotype are shown in tables 1 and 2.
TABLE 1 statistical Table of the distribution differences of alleles between two groups
| Alleles | Susceptible group (%) | Anti-disease group (%) | Chi-square value | P value |
| G | 80(0.25) | 136(0.85) | 18.0528 | 0.0035 |
| T | 66(0.75) | 24(0.15) |
TABLE 2 statistical Table of the distribution differences of genotypes between two groups
| Alleles | Susceptible group (%) | Anti-disease group (%) | Chi-square value | P value |
| GG | 14(0.175) | 56(0.7) | 16.7211 | 0.0027 |
| GT | 66(0.825) | 24(0.3) |
As can be seen from tables 1 and 2, at this site, there was a very significant difference (P <0.01) in both allele and genotype frequencies in the disease-resistant and susceptible populations, with the frequencies of alleles G and T being 85% and 15%, respectively, and the genotype frequencies of genotype GG and genotype GT being 70% and 30%, respectively. Further proves that the 342 th base G or T of the nucleotide sequence (full length 545bp) shown in SEQ ID NO.1 from the 5' end is obviously related to the large yellow croaker Vibrio harveyi disease and is an SNP marker related to the large yellow croaker disease, and the SNP marker can be used for breeding the disease-resistant character of the large yellow croaker.
Sequence listing
<110> Zhejiang ocean university
<120> SNP marker associated with Vibrio harveyi disease of large yellow croaker, primer and application thereof
<140> 2018112174341
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<170> SIPOSequenceListing 1.0
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<213> Pseudosciaena crocea Vibrio harveyi associated SNP marker (SNP markers associated with Harm's disease in large yellow river croaker)
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gttatgctgg atggaaagac ccacatcctg agggtgaacc cacatgacac agtgggctct 120
ctgaaaacgc tcatccagaa caaagtggga gttccccctc agaaacaaaa gctgatcttt 180
gtgaacggac agaggacacc tctcagcgac gactcccagt atatcagcca ctacggtctg 240
cagtccggct ctcaggtgtc tctgctgatc acacagccca agaccttcca ggtgttcctg 300
aacaacctgc agggtcagaa gagcacctat gatatcaaac ckgacgagac tgtgatgaac 360
ttcaagagga gggtggagca aagagagggg gtccctgtga accagcagag gctgctccac 420
caaagcaggg agctgctgga tgggaaactt tcagactacg acgtcaagga actgagcacc 480
atcaacatga caggccgcct gagaggaggt taaaatgtca gaaagataaa atagtatact 540
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<213> Primer-R (Primer-R)
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ttgatggtgc tcagttcctt g 21
Claims (1)
1. The application of the primer pair of the SNP marker in preparing the kit for detecting the susceptibility of the large yellow croaker to the vibrio harveyi disease is characterized in that the nucleotide sequence of the primer pair of the SNP marker is shown in SEQ ID NO. 2-3;
the SNP marker nucleotide sequence is shown as SEQ ID NO.1, and the 342 th base from the 5' end of the sequence is G or T;
the large yellow croaker with the individual genotype of the SNP marker GT is more susceptible to vibrio harveyi.
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| CN110699466A (en) * | 2019-11-18 | 2020-01-17 | 上海海洋大学 | SNP locus associated with large yellow croaker anti-vibrio anguillarum, screening method and application thereof |
| CN111662987A (en) * | 2020-05-21 | 2020-09-15 | 华中农业大学 | Pelteobagrus fulvidraco red head disease resistance related SNP marker, screening method and application |
| CN112501317B (en) * | 2020-12-28 | 2022-10-04 | 厦门大学 | SNP (Single nucleotide polymorphism) markers applicable to Cryptocaryon irritans resistant breeding of large yellow croakers |
| CN117265147B (en) * | 2023-10-08 | 2024-05-03 | 三门县水产技术推广站 | Molecular marker related to vibrio resistance of blood clam |
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| CN105349675A (en) * | 2015-12-02 | 2016-02-24 | 集美大学 | Larimichthys crocea genome-wide SNP and InDel molecular marker method based on double enzyme digestion |
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