SNP (Single Nucleotide Polymorphism) molecular marker combination related to disease resistance of ricefield eel and application thereof

Abstract

The invention discloses an SNP molecular marker combination related to disease resistance of ricefield eels and application of the SNP molecular marker combination, and relates to the technical field of molecular biology. The SNP molecular marker combination comprises the following molecular markers (1)-(3): (1) a molecular marker with a nucleotide sequence as shown in SEQ ID NO.7; (2) a molecular marker with a nucleotide sequence as shown in SEQ ID NO.8; and (3) a molecular marker with a nucleotide sequence as shown in SEQ ID NO.9. According to the invention, an SNP locus in a TLR7 gene in a monopterus albus group is screened through sequence alignment, and genotyping is carried out on the SNP locus; and comparing the differences of the disease resistance of the genotypes, and screening to obtain the genotype with the strongest disease resistance, thereby providing an effective molecular marker for detecting the disease resistance of the ricefield eel. The molecular marker combination provided by the invention is associated with the disease resistance of the monopterus albus, and provides effective technical support for breeding of disease-resistant monopterus albus varieties.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of molecular biology, in particular to a SNP molecular marker combination related to the disease resistance of Monopterus albus and application thereof. BACKGROUND

[0002] Monopterus albus, commonly known as eel, belongs to Chordata, Osteichthyes, Synbranchiformes, Synbranchidae and Monopterus. Monopterus albus has tender and nutritious meat, and the content of high-quality protein in its muscle is up to 18%-20%. Monopterus albus is rich in DHA, vitamins, calcium, iron and other minerals, and has both edible value and traditional medicinal value (it is believed in traditional Chinese medicine that it can tonify qi and blood and benefit spleen and stomach). Under this background, the artificial breeding scale of Monopterus albus is expanding, and the breeding mode is gradually transforming from traditional ecological breeding in rice fields to intensive breeding in ponds and factory-like recirculating water breeding. However, with the increase of breeding density and the deepening of intensification, Monopterus albus breeding industry is facing prominent problems: deterioration of breeding environment (accumulation of residual feed and excretion leads to eutrophication of water body, ammonia nitrogen and nitrite exceed standard), and intensified stress response, which leads to frequent occurrence of diseases, including saprolegniasis, red skin disease, enteritis and edwardsiella disease, etc. The incidence of disease can reach 30%-50%, and the mortality rate can exceed 70% in severe cases. At present, there are obvious shortcomings in the immune regulation mechanism, pathogen identification, diagnosis technology and new variety breeding of Monopterus albus.

[0003] Therefore, systematic mining of Monopterus albus disease resistance genes, analysis of its immune defense mechanism, development of efficient disease diagnosis and control technology, and breeding of disease-resistant high-yield excellent varieties are the key content of current Monopterus albus aquaculture research field, which has important theoretical and practical significance for the sustainable development of aquaculture industry.

[0004] Single nucleotide polymorphism (SNP) is a DNA sequence polymorphism caused by a single nucleotide variation at the genome level, which is the most common type of heritable variation. It exists widely in various organisms and has high genetic stability. In recent years, SNP markers have also become one of the mainstream breeding methods. This breeding method has achieved good research results in crops and large livestock. The application of SNP breeding in aquaculture is one of the current research focuses. Moreover, the development of sequencing technology in recent years also provides a good foundation for SNP breeding. The establishment of gene library using sequencing technology and the screening of excellent disease-resistant alleles based on the gene library provide a new idea for Monopterus albus disease-resistant variety breeding by combining sequencing technology and SNP markers. SUMMARY

[0005] The application aims to provide a SNP molecular marker combination related to the disease resistance of Monopterus albus and an application thereof, so as to solve the problems in the prior art. The molecular marker combination provided by the application is related to the disease resistance of Monopterus albus and can be applied to the identification of the disease resistance of Monopterus albus, thereby providing effective technical support for the breeding of Monopterus albus varieties with disease resistance.

[0006] To achieve the above-mentioned object, the application provides the following solutions.

[0007] The application provides a SNP molecular marker combination related to the disease resistance of Monopterus albus, which comprises the following (1)-(3) molecular markers.

[0008] (1) a molecular marker with a nucleotide sequence as shown in SEQ ID NO. 7, wherein SNP sites exist at positions 904, 1045 and 1048 of the nucleotide sequence, and the SNP sites are C / T, C / G and G / A mutations, respectively.

[0009] (2) a molecular marker with a nucleotide sequence as shown in SEQ ID NO. 8, wherein a SNP site exists at position 275 of the nucleotide sequence, and the SNP site is a C / G mutation.

[0010] (3) a molecular marker with a nucleotide sequence as shown in SEQ ID NO. 9, wherein SNP sites exist at positions 466 and 823 of the nucleotide sequence, and the SNP sites are A / C and A / G mutations, respectively.

[0011] The application further provides a primer combination for detecting the disease resistance of Monopterus albus, wherein the primer combination comprises the following (a)-(c) primers.

[0012] (a) an upstream primer TLR7-F1 with a nucleotide sequence as shown in SEQ ID NO. 1 and a downstream primer TLR7-R1 with a nucleotide sequence as shown in SEQ ID NO. 2;

[0013] (b) an upstream primer TLR7-F2 with a nucleotide sequence as shown in SEQ ID NO. 3 and a downstream primer TLR7-R2 with a nucleotide sequence as shown in SEQ ID NO. 4;

[0014] (c) an upstream primer TLR7-F3 with a nucleotide sequence as shown in SEQ ID NO. 5 and a downstream primer TLR7-R3 with a nucleotide sequence as shown in SEQ ID NO. 6.

[0015] The application further provides an application of the above-mentioned molecular marker combination in detecting the disease resistance of Monopterus albus for non-disease diagnosis or treatment purposes.

[0016] Further, the disease resistance of Monopterus albus refers to the disease resistance of Monopterus albus to rhabdovirus.

[0017] The application also provides application of the primer combination in the preparation of a detection product for detecting the disease resistance of the Misgurnus anguillicus.

[0018] Further, the disease resistance of the Misgurnus anguillicus refers to the disease resistance of the Misgurnus anguillicus to rhabdovirus.

[0019] The application also provides a detection product for detecting the disease resistance of the Misgurnus anguillicus, comprising the primer combination.

[0020] The application also provides application of the primer combination or the detection product in detecting the disease resistance of the Misgurnus anguillicus for non-disease diagnosis or treatment purposes.

[0021] Further, the disease resistance of the Misgurnus anguillicus refers to the disease resistance of the Misgurnus anguillicus to rhabdovirus.

[0022] The application also provides a method for detecting the disease resistance of the Misgurnus anguillicus for non-disease diagnosis or treatment purposes, comprising the following steps:

[0023] Extracting blood lymph RNA of a to-be-detected Misgurnus anguillicus individual to obtain cDNA through reverse transcription;

[0024] Using the cDNA as a template, performing PCR amplification on the primer combination, obtaining a genotype through sequencing, and judging the disease resistance of the to-be-detected Misgurnus anguillicus individual: the individual with the CCACCG haplotype has stronger resistance to rhabdovirus than other haplotypes.

[0025] The application discloses the following technical effects:

[0026] The application screens the SNP site in the TLR7 gene in the Misgurnus anguillicus population through sequence alignment, and performs genotyping on the SNP site; the differences in disease resistance between different genotypes are compared, and the genotype with the strongest disease resistance is screened, thereby providing an effective molecular marker for detecting the disease resistance of the Misgurnus anguillicus. The screening method for the disease resistance of the Misgurnus anguillicus based on the molecular marker can screen the Misgurnus anguillicus with excellent disease resistance by using the SNP difference of the disease resistance gene, and then be used for disease resistance breeding. The application provides effective technical support for breeding of the Misgurnus anguillicus with disease resistance. BRIEF DESCRIPTION OF DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed in the embodiments. Obviously, the drawings described below only show some embodiments of the application, and those skilled in the art can also obtain other drawings according to these drawings without creative effort.

[0028] Figure 1 is a differential expression gene column chart, wherein the blue color represents up-regulated expression, and the gray color represents down-regulated expression;

[0029] Figure 2 Figure for transcriptome credibility verification result;

[0030] Figure 3 Figure for detection result of TLR7 gene tissue-specific expression in Monopterus albus;

[0031] Figure 4 Figure for division of Monopterus albus anti-disease ability difference groups; wherein, A is a survival rate statistical chart of three times of anti-disease ability verification; B is a single-day death number statistical chart of three times of anti-disease ability verification;

[0032] Figure 5 Figure for correlation analysis of different haplotypes of TLR7 gene in three times of anti-disease ability verification. DETAILED DESCRIPTION

[0033] Various exemplary embodiments of the present application will now be described in detail, which should be considered to be illustrative of the application and not restrictive of the application. It should be understood that the detailed description and specific examples, while indicating certain aspects of the application, are given by way of illustration only, since various changes and modifications within the scope of the application will become apparent to those skilled in the art from this detailed description.

[0034] It should be understood that the terms used in the specification of the present application are merely used to describe particular embodiments and are not intended to limit the present application. In addition, for numerical ranges in the present application, it should be understood that each intermediate value between the upper limit and the lower limit of the range is specifically disclosed. Each intermediate value within any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the present application. The upper limit and the lower limit of these smaller ranges can be included or excluded independently.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials in connection with which the documents are cited. In case of conflict between the content of the specification and that of any document incorporated herein by reference, the content of the specification prevails.

[0036] Various modifications and changes can be made to the specific embodiments of the present application described herein without departing from the scope or spirit of the application. Other embodiments of the application will be apparent to those of ordinary skill in the art from the description and examples presented herein. The description and examples are illustrative of the application and are not intended to limit the scope of the application.

[0037] As used herein, the terms "comprise", "comprising", "include", "including", "have", "having" and the like are open-ended and do not exclude additional, unrecited elements or method steps.

[0038] Example 1

[0039] According to the transcriptome information, a new disease-resistant gene Toll-like receptor 7 (TLR7) gene is screened, and the SNP haplotype thereof is analyzed for disease resistance, as follows:

[0040] 1. Disease-resistant gene and SNP site mining

[0041] (1) Extraction of blood lymph RNA of healthy rice eels and rice eels stimulated by CrERV for 24 h and 48 h, with the specific method as follows:

[0042] The rhabdovirus (Chinese rice-field eel rhabdovirus, CrERV) is diluted with physiological saline to a final concentration of 10 6 copies / mL, and 200 μL of the rhabdovirus diluent is injected into the abdominal cavity of each rice eel. Healthy rice eels are injected with an equal volume of physiological saline. After 24 h and 48 h of cultivation under laboratory conditions, blood lymph RNA extraction is performed, with the specific method as follows:

[0043] 150 μL of blood sample is extracted from the rice eel, placed in an EP tube, and placed on ice. 200 μL of pre-cooled Trizol reagent (purchased from Baosheng Engineering Dalian Co., Ltd.) is added, and the Trizol solution (reagent) is ground with a grinder until it is pink. 600 μL of Trizol reagent is added to each sample again; after standing at room temperature for 5 min, centrifugation is performed at 4°C and 12000×g for 10 min; 900 μL of supernatant is taken and placed in a new EP tube, and 200 μL of chloroform is added, which is mixed well under shaking conditions without vortex shaking, and shaken for about 15 s before and after, and then left to stand at room temperature for 5 min; centrifugation is performed at 4°C and 12000×g for 10 min; after centrifugation, the solution presents three layers, and the upper clear liquid is carefully taken with a gun head below the liquid surface, 400 μL of isopropanol is added to 400 μL, and gently mixed, and left to stand at room temperature for 5 min; centrifugation is performed at 4°C and 12000×g for 15 min until a white precipitate is observed; the supernatant is removed, and the precipitate is not aspirated, and if there is no precipitate, a small amount of solution is left, 1 mL of 75% ethanol prepared with DEPC water is added, and resuspended; centrifugation is performed at 4°C and 8000×g for 5 min, and the supernatant is removed; the solution is aspirated dry, and the EP tube is placed in a fume hood to dry for 5 min.

[0044] (2) The RNA is sent to a commercial sequencing company (Shanghai Meiji Biomedicine Technology Co., Ltd.) for transcriptome library construction, sequence assembly, and differential expression gene (DEG) analysis.

[0045] (3) According to the results of differential expression gene analysis, the target gene is preliminarily screened. The differential expression gene analysis is shown in Figure 1 .

[0046] (4) The primers were designed to verify the expression of DEGs by using fluorescent quantitative PCR. The qRT-PCR primer sequences are shown in Table 1. The verification results are shown in Figure 2 .

[0047] Table 1 qRT-PCR primer sequences

[0048]

[0049] (5) The previous qRT-PCR primers were used to detect the tissue specificity of each tissue of the rice field eel. The RNA extraction method was the same as before. The cDNA was obtained by reverse transcription using the reverse transcription kit produced by Tiangen Biochemical (Beijing) Technology Co., Ltd. The specific steps are as follows: 8 μL of RNA solution, 2 μL of 5×FastKing-RT SuperMix, 10 μL of ddH2O were mixed in a PCR tube, and then placed in a PCR instrument. The reaction program was as follows: 42℃ for 15 min; 95℃ for 3 min. The tissue specificity detection results are shown in Figure 3 .

[0050] (6) The sub-segment amplification primers of the full-length sequence were designed according to the transcriptome splicing results. The primer sequences are shown in Table 2.

[0051] Table 2 Full-length sequence amplification primers

[0052]

[0053] The nucleotide sequence of the amplified fragment 1 is shown in SEQ ID NO. 7. There are SNP sites at the 904th, 1045th and 1048th bases, which are C / T, C / G and G / A mutations, respectively, and are recorded as SNP1, SNP2 and SNP3.

[0054]

[0055] The nucleotide sequence of the amplified fragment 2 is shown as SEQ ID NO. 8, and a SNP site exists at the 275th base, which is a C / G mutation, denoted as SNP4.

[0056]

[0057] The nucleotide sequence of the amplified fragment 3 is shown as SEQ ID NO. 9, and there are SNP sites at the 466th and 823rd bases, which are A / C and A / G mutations, respectively, and are denoted as SNP5 and SNP6, respectively.

[0058]

[0059] The segment primer was used to amplify the target fragment, and the fragment integrity was detected by agarose gel electrophoresis. The PCR conditions were as follows: 95 °C pre-denaturation for 5 min; 95 °C denaturation for 30 s, 60 °C annealing for 30 s, 72 °C extension for 30 s, 35 cycles; 72 °C re-extension for 10 min; 4 °C storage.

[0060] (7) The RNAs of 300 individual rice eels were extracted and detected, respectively, and the cDNAs were obtained after reverse transcription and used as templates for PCR amplification, and the specific steps were as follows:

[0061] The segment primer was used to amplify the target fragment, and the fragment integrity was detected by agarose gel electrophoresis. The PCR conditions were as follows: 95 °C pre-denaturation for 5 min; 95 °C denaturation for 30 s, 60 °C annealing for 30 s, 72 °C extension for 30 s, 35 cycles; 72 °C re-extension for 10 min; 4 °C storage.

[0062] (8) The PCR products were sent to a commercial sequencing company (Shanghai Sangon Biological Technology Co., Ltd.) for sequencing, and after the sequencing company fed back the specific sequence information, the software Sequencher was used to compare the sequence differences of the disease-resistant genes among the individuals, and the SNP sites were screened. The screening results are shown in Table 3.

[0063] Table 3 Summary of TLR7 gene SNP typing results

[0064]

[0065] 2. Detection of the disease resistance of rice eels

[0066] (1) 120 rice eels were taken, 200 μL of blood lymph was taken from each rice eel, RNA was extracted, and cDNA was reverse transcribed, and then PCR amplification was performed to detect the genotypes of 6 SNP sites, and the primers, reaction system and reaction conditions were the same as those in step (6) of “1. Disease-resistant gene and SNP site mining”. Then each rice eel was labeled and inoculated with rhabdovirus, and the specific inoculation method was the same as before. The survival time of each rice eel was observed and recorded. Repeat three times, and draw into Figure 4 .

[0067] (2) The genotypes of TLR7 gene SNP sites were corresponded with the survival time. According to the survival time, the disease resistance differences of different genotypes were screened. Through induction and summary, the strongest genotype combination of disease resistance was obtained (see Figure 5 ).

[0068] (3) Result analysis: In the three disease resistance experiments, the average survival time was between 72-144 hours, therefore, survival time less than 72 hours was poor disease resistance; survival time greater than 144 hours was strong disease resistance. The results showed that the survival time of the TLR7-2 haplotype (i.e. CCACCG) of the rice field eel was in the strong disease resistance proportion the most in the three experiments, and the disease resistance was the strongest.

[0069] The above-described embodiments are merely preferred modes of the present application, and are not intended to limit the scope of the present application. Various modifications and improvements to the technical solutions of the present application made by those skilled in the art without departing from the design spirit of the present application shall fall within the protection scope of the present application as defined by the claims.

Claims

1. A combination of SNP molecular markers related to the disease resistance of swamp eels, characterized in that, Including the molecular markers described in (1)-(3) below: (1) The molecular marker with the nucleotide sequence shown in SEQ ID NO.7 has SNP sites at bases 904, 1045, and 1048, which are C / T, C / G, and G / A mutations, respectively. (2) The molecular marker with a nucleotide sequence as shown in SEQ ID NO.8 has an SNP site at the 275th base, which is a C / G mutation; (3) The molecular marker with nucleotide sequence as shown in SEQ ID NO.9 has SNP sites at bases 466 and 823, which are A / C and A / G mutations, respectively.

2. A primer combination for detecting disease resistance in swamp eels, characterized in that, The primer combination includes the primers described in (a)-(c) below: (a) The upstream primer TLR7-F1 with the nucleotide sequence shown in SEQ ID NO.1 and the downstream primer TLR7-R1 with the nucleotide sequence shown in SEQ ID NO.2; (b) The upstream primer TLR7-F2 with the nucleotide sequence shown in SEQ ID NO.3 and the downstream primer TLR7-R2 with the nucleotide sequence shown in SEQ ID NO.4; (c) The upstream primer TLR7-F3 with the nucleotide sequence shown in SEQ ID NO.5 and the downstream primer TLR7-R3 with the nucleotide sequence shown in SEQ ID NO.

6.

3. The application of the molecular marker combination as described in claim 1 in detecting the disease resistance of swamp eels for non-disease diagnosis or treatment purposes.

4. The application according to claim 3, characterized in that, The disease resistance of the eel refers to its resistance to rhabdoviruses.

5. The application of the primer combination as described in claim 2 in the preparation of a detection product for the disease resistance of swamp eels.

6. The application according to claim 5, characterized in that, The disease resistance of the eel refers to its resistance to rhabdoviruses.

7. A product for detecting the disease resistance of eels, characterized in that, Includes the primer combination described in claim 2.

8. The use of a primer combination as described in claim 2 or a detection product as described in claim 7 in detecting the disease resistance of swamp eels for non-disease diagnosis or treatment purposes.

9. The application according to claim 8, characterized in that, The disease resistance of the eel refers to its resistance to rhabdoviruses.

10. A method for detecting the disease resistance of swamp eels for non-disease diagnosis or treatment purposes, characterized in that, Includes the following steps: Hemolymph RNA was extracted from individual eels to be tested and reverse transcribed to obtain cDNA; Using the cDNA as a template, PCR amplification was performed using the primer combination described in claim 2. The genotype was obtained by sequencing, and the disease resistance of the tested eel individuals was determined: individuals with the CCACCG haplotype showed stronger resistance to rhabdovirus than other haplotypes.

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