SNP (Single Nucleotide Polymorphism) marker for identifying vibrio-resistant tegillarca granosa and specific primer and application thereof

Abstract

The invention discloses an SNP (Single Nucleotide Polymorphism) marker for identifying vibrio-resistant tegillarca granosa as well as a specific primer and application thereof, belongs to the technical field of aquaculture genetic breeding marker screening, and discloses a combination of six SNP loci for identifying vibrio-resistant tegillarca granosa. The SNP marker combination and the identification method disclosed by the invention can be used for molecular marker-assisted breeding of vibrio-resistant tegillarca granosa, and the blank that no SNP site can be used for identifying the vibrio-resistant tegillarca granosa is filled. The invention provides a powerful technical support for breeding tegillarca granosa with excellent vibrio resistance.

Description

Technical Field

[0001] The present invention belongs to the technical field of screening genetic breeding markers for aquaculture, and particularly relates to an SNP marker for identifying Vibrio-resistant blood clams, a specific primer thereof, and applications thereof. Background Art

[0002] The blood clam (Tegillarca granosa) belongs to the phylum Mollusca, class Bivalvia, order Taxodonta, family Arcidae, and genus Tegillarca. It is a traditional cultured shellfish in China. Local names: granular clam, bloody clam, blood snail, tile ridge clam. It is distributed along the coasts of China. The spawning period from August to October is the peak production season. In addition, artificial cultivation is carried out in Hebei, Shandong, Zhejiang, Fujian, and Guangdong, with a high yield. The meat of the blood clam is delicious, and can be eaten fresh or pickled with wine, or made into dried products. The clam meat contains a large amount of protein and vitamin (B12). The clam blood is bright red, and there is a golden thread-like color line along the edge of the meat. The shell can be used as medicine, with the effects of dissipating blood clots and resolving phlegm accumulation. Because of its low price, it can be regarded as a popular seafood product.

[0003] Since 2000, epidemic diseases and large-scale deaths of cultured blood clams have occurred from time to time. Among them, Vibrio diseases are the most common and cause the most serious deaths, which have become one of the bottleneck problems restricting the healthy development of the blood clam aquaculture industry. Vibrios are widely distributed in the sea water of estuaries, bays, and coastal waters and in marine animals. The main pathogenic bacteria include Vibrio parahaemolyticus, Vibrio harveyi, Vibrio anguillarum, Vibrio vulnificus, Vibrio alginolyticus, etc. Therefore, starting from the germplasm level, deeply exploring and utilizing the Vibrio-resistant germplasm of blood clams, breeding excellent new varieties with strong antibacterial ability, and screening seedlings with strong antibacterial ability for aquaculture production have become the key points and hotspots in the research and development of bacterial disease prevention and control.

[0004] Single nucleotide polymorphism (SNP) refers to DNA sequence polymorphisms caused by single-base variations at the genomic level. Such variations are mainly caused by single-base transitions and transversions. They are extremely numerous, rich in polymorphisms, have dimorphism, are easy to detect and statistically analyze, and can achieve high-throughput automated detection (Durstewitz G et al, 2010). Currently, SNPs, together with simple sequence repeat (SSR) molecular markers, are the only two recommended marker methods in the DNA fingerprint database construction in the BMT molecular test guidelines of the International Union for the Protection of New Varieties of Plants (UPOV) and the General Rules for DNA Fingerprinting Methods for Plant Variety Identification (NY / T 2594-2016) in China. SSR molecular markers have certain limitations due to problems such as limited marker numbers, difficulties in integrating detection data, and time-consuming operations, while SNP molecular markers can make up for these defects based on their own advantages.

[0005] In the breeding and genetic breeding of aquatic animal seedlings, SNP markers have been used for many years, and some molecular markers related to growth, reproduction, disease resistance traits, etc. have been successively screened. The tetra-primer amplification refractory mutation system PCR is a new SNP detection method. This method only needs to design four specific primer pairs to perform PCR amplification on the template to obtain the genotyping of an SNP locus. The tetra-primer amplification refractory mutation system PCR not only has the accuracy of DNA sequencing but also overcomes the disadvantages of high cost, cumbersome operation, false positives, etc., and has no special requirements for the detected sequence sites. Therefore, it is favored in breeding work and has a very broad application prospect. Summary of the Invention

[0006] Aiming at the deficiencies of the existing technology, the purpose of the present invention is to provide specific SNP markers for identifying Vibrio-resistant blood clams to solve the problems existing in the above-mentioned existing technology. By detecting the unique SNP markers of Vibrio-resistant blood clams, the accurate identification of Vibrio-resistant blood clams is realized.

[0007] To achieve the above purpose, the present invention provides the following technical solutions:

[0008] The present invention provides a specific SNP marker for identifying Vibrio-resistant blood clams, and the SNP molecular marker includes any one or a combination of at least two of the following 6 SNP molecular markers:

[0009]

[0010] The nucleotide sequence of chromosome TgAP-1 is as shown in SEQ ID NO.1.

[0011] Preferably, the vibrio-resistant blood clam is a Vibrio harveyi-resistant blood clam.

[0012] The present invention also provides a primer set for identifying vibrio-resistant blood clams, and the primer set includes primer TgAP-1-F and primer TgAP-1-R for detecting the SNP markers described in claim 1.

[0013] The nucleotide sequence of the primer TgAP-1-F is as shown in SEQ ID NO.2;

[0014] The nucleotide sequence of the primer TgAP-1-R is as shown in SEQ ID NO.3.

[0015] The present invention also provides a kit for identifying vibrio-resistant blood clams, and the kit includes a primer set with nucleotide sequences as shown in SEQ ID NO.2 to SEQ ID NO.3.

[0016] Preferably, the kit further includes a reagent for extracting genomic DNA from a sample and a reagent for performing a PCR reaction using the primer set with nucleotide sequences as shown in SEQ ID NO.2 to SEQ ID NO.3.

[0017] In another aspect of the present invention, there is also provided the use of the SNP markers or primer combinations or kits described above in identifying vibrio-resistant blood clams.

[0018] In another aspect of the present invention, there is also provided a method for identifying vibrio-resistant blood clams, and the method includes the following steps:

[0019] 1) Extract the DNA of the blood clam to be tested, detect its quality and concentration, and dilute it to a uniform working concentration;

[0020] 2) Using the DNA in step (1) as a template, add specific primers for detecting the SNP locus combination as described above and a PCR premix, and perform PCR amplification respectively. The amplification products are subjected to Sanger sequencing. If the sequencing results of the PCR products of the blood clam to be tested are consistent with at least one of the above 6 SNP molecular markers, then the blood clam to be tested is a vibrio-resistant blood clam; the specific primers are composed of the detection primers for the above 6 SNP loci, and the sequences of the specific primers for detecting each SNP locus are as shown in SEQ ID NO.2 to SEQ ID NO.3.

[0021] Preferably, extract the DNA from the parts of the blood clam other than the hepatopancreas.

[0022] The beneficial effects of the present invention are:

[0023] Based on the whole-genome resequencing data of representative materials of Vibrio-resistant blood clams, the seqMan software was used to splice and analyze the sequencing results, potential SNP sites were searched, and 6 SNP sites suitable for the identification of Vibrio-resistant blood clams were screened out. The 6 specific SNP sites provided by the present invention were determined through multi-sample tests, with good stability and easy to promote and apply, filling the blank of specific identification markers for Vibrio-resistant blood clams, and 2 primer sequences for detecting SNP sites were designed. Using these 2 primer sequences, effective high-throughput and high-efficiency purity identification of 'Vibrio-resistant blood clams' can be achieved, which can be used to screen blood clam individuals with Vibrio resistance as breeding parents, thus providing strong technical support for the genetic breeding of blood clams. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 : Detection and genotyping peak maps of TgSNP-1 (a), TgSNP-2 (b), TgSNP-4 (c), and TgSNP-7 (d). DETAILED DESCRIPTION OF THE EMBODIMENTS

[0025] The present invention will be further described in detail below with reference to examples and effect examples, rather than limiting the scope of the present invention.

[0026] Example 1: Screening of Vibrio harveyi-resistant blood clam populations and susceptible populations

[0027] The Vibrio-resistant blood clam populations (09-R), common control blood clam populations (09-C), Vibrio-resistant families (17-R), and Vibrio-susceptible families (17-S) used in this experiment were all independently cultivated by the Zhejiang Institute of Marine Fisheries (Wenzhou, Zhejiang).

[0028] In 2009, about 1000 adult natural blood clam populations were collected from Yueqing Bay, Wenzhou City, Zhejiang Province (121°E, 28°N). After one week of temporary cultivation and temperature control, they were placed in a 2000L fiberglass cylindrical culture barrel, and Vibrio harveyi (1×10 7(CFU / mL). The sewage was cleaned once a day, and Isochrysis galbana was fed regularly twice a day, in the morning and evening. The clean sand-filtered seawater was replaced every morning, and fresh Vibrio was added until more than 30% of the blood clams died, then the virus challenge was stopped. The remaining blood clams were transferred to another disinfected culture bucket and fed normally to allow the blood clams to recover on their own. Dead blood clams were removed in a timely manner every day until no blood clams died for a continuous week. The blood clams that survived the virus challenge experiment were preserved in the laboratory and allowed to reproduce naturally, obtaining the first-generation resistant blood clam population 09-R with Vibrio resistance. During the same period, the offspring of the blood clam population that was not tested for virus challenge were used as the control population 09-C. The 09-C and 09-R populations were raised in the same environment, and every 2-3 years, they were allowed to reproduce naturally to produce corresponding offspring populations. In this experiment, the fifth-generation blood clam population cultivated in 2021 was used.

[0029] In addition, in the summer of 2017, adult blood clams with well-developed gonads were collected from Yueqing Bay. After being exposed to air for 8 h, they were induced to spawn. According to the male and female genders, the blood clams were selected and placed in different beakers. According to the nested half-sib mating principle, each male blood clam was mated with 2-3 different male and female blood clams, and a total of 16 half-sib families and 46 full-sib families were produced. In 2019, 5520 blood clams from 46 full-sib families were used for Vibrio harveyi virus challenge detection. Twenty temporarily cultured blood clams were randomly taken out from 09-R and 09-C respectively, and divided into a resistant virus challenge group (n = 10), a resistant control group (n = 10), a common virus challenge group (n = 10), and a common control group (n = 10). The virus challenge concentration set in the experiment was 1×10 7 CFU / mL, and the virus challenge temperature was 28.0 ± 0.5 °C. The sand-filtered seawater was changed regularly once a day during the virus challenge, and Vibrio was re-added after feeding Navicula minima. After 24 h of infection, the gill tissues of 5 blood clams were dissected from each group and stored at -80 °C for analyzing the expression pattern of TgAP-1 in different resistant populations after infection. In the control group set in the experiment, Vibrio was not added, and other treatments were the same as those in the virus challenge group. According to the survival differences after virus challenge, 4 families with higher survival rates were selected to form the resistant blood clam family 17-R, and 4 families with higher mortality rates were selected to form the susceptible blood clam family 17-S.

[0030] 2) Preparation of the fermented bacterial liquid of Vibrio harveyi

[0031] The Vibrio harveyi strain was streaked on an LB solid medium. After culturing at 27 °C for 18 h, single colonies were picked and inoculated into 100 mL of 2216E liquid medium, and cultured in a shaker at 150 r / min and 27 °C for 24 h to prepare the seed liquid. The seed liquid was then added to a large volume of 2216E liquid medium at a ratio of 1:100 for culture. After culturing for 24 h, the fermented bacterial cells of Vibrio harveyi were obtained by centrifugation.

[0032] Example 2: Development of Specific SNP Markers for Arcobacter-Resistant Blood Clams

[0033] Specifically, it includes the following steps:

[0034] Use a DNA extraction kit to extract the genomic DNA of blood clams. The specific steps are as follows:

[0035] ① Cut about 30 mg of the adductor muscle of blood clams, cut it into pieces, put it into 200 μL of tissue cell lysate, and add an appropriate amount of disruption beads.

[0036] ② Add 20 μL of proteinase K, vortex and mix well, and place it in a crusher to crush for 20 - 30 s.

[0037] ③ Place it at 56 °C for 2 h, and shake and mix the sample 2 - 3 times every 30 min.

[0038] ④ Add 200 μL of buffer GB, vortex and mix well, then place it at 70 °C for 10 min, and take it out after the solution becomes clear.

[0039] ⑤ Add 200 μL of absolute ethanol, vortex and mix well the flocculent precipitate that appears, and briefly centrifuge to remove the liquid on the tube wall and lid.

[0040] ⑥ Add the obtained solution to a DNA adsorption column, centrifuge at 12000 rpm for 30 sec, and discard the waste liquid.

[0041] ⑦ Add 500 μL of buffer GD to the adsorption column, centrifuge at 12000 rpm for 30 sec, and discard the waste liquid.

[0042] ⑧ Add 600 μL of washing buffer PW to the adsorption column, centrifuge at 12000 rpm for 30 sec, and discard the waste liquid. Repeat once.

[0043] ⑨ Centrifuge at 12000 rpm for 2 min, discard the waste liquid, and place it at room temperature for several minutes to thoroughly dry the washing buffer.

[0044] ⑩ Put the adsorption column into a clean centrifuge tube, slowly and dropwise add 50 μL of elution buffer TE, place it at room temperature for 2 - 5 min, centrifuge at 12000 rpm for 2 min, and collect the DNA solution.

[0045] Randomly take out the TgAP-1 gene fragments of 20 blood clams in the 09-C population, mix them in equal proportion to form a DNA template, and obtain the gene sequence of the mixed sample TgAP-1 by direct sequencing. Use the seqMan software to splice and analyze the sequencing results to find potential SNP sites.

[0046] Specifically, taking the untranslated region and coding region of the TgAP-1 gene as the main targets, potential SNP sites were searched. To further improve the accuracy of the sequencing results, two pairs of amplification primers were designed in this application. The nucleotide sequence of primer sTgAP-1-1 is shown in SEQ ID NO.4-5, and the nucleotide sequence of primer sTgAP-1-2 is shown in SEQ ID NO.6-7. The TgAP-1 gene was divided into two sequences with repetitive base fragments. Using the genomic DNA of 75 Tegillarca granosa of 09-C as a template, PCR amplification was performed. After detecting the quality of the amplified PCR products by agarose gel electrophoresis, they were sent to Sangon Biotech (Shanghai) Co., Ltd. for bidirectional sequencing. The sequencing results were assembled using the seqMan subroutine of DNAstar. Base deletions were default ignored, and only base site changes were used as the criterion for judging the existence of potential SNPs. Genotyping was performed according to the sequencing chromatograms. The discrimination criterion was that when there were overlapping peaks, if the height of one absorption peak exceeded 50% of the other absorption peak, it was recognized as a double-peak site, that is, an SNP site. The minor allele frequency (MAF) was calculated using Popgene 32 software and the Hardy-Weinberg equilibrium (HWE) was tested. SNP sites with MAF < 5% or deviating from HWE (P < 0.05) were excluded from subsequent association analyses. The results are as Figure 1 shown. A total of 18 SNPs were identified from the 09-C Tegillarca granosa population that conformed to the HWE equilibrium and had MAF ≥ 0.05. According to the genotyping results in the 09-C population, there was an A > G mutant SNP site, TgSNP-1, in the 5’UTR; there were 6 mutant SNP sites in the CDS region, namely TgSNP-2 (G > A), TgSNP-3 (T > C), TgSNP-4 (T > G), TgSNP-5 (A > C), TgSNP-6 (C > A), and TgSNP-7 (C > T); and there were 9 mutant SNP sites in the 3’UTR, namely TgSNP-8 (T > C), TgSNP-9 (A > T), TgSNP-10 (T > A), TgSNP-11 (A > G), TgSNP-12 (G > A), TgSNP-13 (A > T), TgSNP-14 (C > A), TgSNP-15 (A > T), TgSNP-16 (T > C), TgSNP-17 (A > T), and TgSNP-18 (C > T).

[0047] Eighty blood cockles were randomly selected from the resistant blood cockle population 09-R and the common blood cockle population 09-C respectively. Using the genome of each blood cockle individual as a template and primers shown in SEQ ID NO.2-3, amplification and sequencing were carried out one by one. Taking the detected SNP sites as a reference, an association analysis of Vibrio resistance was conducted in blood cockle populations with different resistances. Pearson's chi-squared test was performed using SPSS 22.0 software. A chi-square value < 0.05 was considered to indicate a significant correlation.

[0048] Table 10 Correlation between SNPs of TgAP-1 gene and Vibrio harveyi resistance in 09-R and 09-C

[0049]

[0050]

[0051]

[0052] To verify the reliability of the screened SNP sites related to Vibrio resistance, 64 blood cockles were randomly selected from the resistant family (17-R) and the susceptible family (17-S) of blood cockles respectively. Using the direct sequencing method for single samples, the differences in SNPs and the association with Vibrio resistance were compared between families with different Vibrio resistances. The results showed that there was a SNP site TgSNP-1 significantly related to Vibrio resistance in the 5’UTR (P < 0.05); there were 3 SNP sites TgSNP-2, TgSNP-4 and TgSNP-7 significantly related to Vibrio resistance in the CDS region (P < 0.05). At the same time, 2 other SNPs (TgSNP-11, TgSNP-12) were also found to be significantly related to Vibrio resistance in the 3’UTR (P < 0.05), and the results are shown in Table 2.

[0053] Table 11 Correlation between SNPs of TgAP-1 gene and Vibrio harveyi resistance in 17-R and 17-S

[0054]

[0055]

[0056]

[0057]

[0058]

[0059] Note: * indicates a significant difference between the 17-R and 17-S populations (P < 0.05).

[0060] Through the SNP analysis of the antagonistic population 09-R and the control population 09-C, it was found that there were 4 SNPs loci related to Vibrio resistance in the TgAP-1 gene, 1 located in the 5'UTR region and 3 SNPs located in the protein coding region. Among the 3 mutation sites in the protein coding region, 2 were synonymous mutations of glutamine (Gln) and threonine (Thr), and 1 was a non-synonymous mutation from leucine (Leu) to tryptophan (Trp). Moreover, these 3 SNPs were all located within the Jun superfamily domain of TgAP-1, and the differences in the Jun domain were exactly the reasons for the diversity of AP-1 among different species. It was speculated that the base mutations in this part would cause functional differences in AP-1. Similarly, in the 17-R resistant family and the 17-S susceptible family with significantly different phenotypes of Vibrio resistance, these 4 SNPs were also found to be significantly associated with Vibrio resistance. In addition, another 2 SNPs significantly correlated with Vibrio resistance were found in the 3'UTR. Non-synonymous mutation SNPs can cause amino acid changes, leading to changes in protein function and biological phenotypes. Although synonymous mutation SNPs do not cause amino acid changes, they can still affect protein function in other ways, such as changing the efficiency of protein translation or affecting mRNA splicing and stability. In addition, there are various gene regulatory factors in the 5'UTR, which play an important role in the translation initiation process. The 3'UTR is mainly responsible for mRNA localization, maintaining mRNA stability, and controlling translation efficiency. The 3'UTR can regulate the formation of protein complexes or post-translational modifications by mediating protein-protein interactions.

[0061] The above content is a further detailed description of the present invention in combination with specific preferred embodiments. It cannot be determined that the specific implementation of the present invention is only limited to these descriptions. For those of ordinary skill in the technical field to which the present invention belongs, without departing from the concept of the present invention, its architecture form can be flexible and variable, and a series of products can be derived. Just making several simple deductions or substitutions should be regarded as belonging to the patent protection scope determined by the claims submitted for the present invention.

Claims

1. A specific SNP marker for identifying Vibrio-resistant blood clams, characterized in that, The SNP molecular marker includes any one or a combination of at least two of the following 6 SNP molecular markers: The nucleotide sequence of chromosome TgAP-1 is shown in SEQ ID NO.

1.

2. The specific SNP marker for identifying Vibrio-resistant blood clams according to claim 1, characterized in that, The vibrio-resistant blood clam is the blood clam resistant to Vibrio harveyi.

3. A primer set for identifying Vibrio-resistant blood clams, characterized in that, The primer set includes primer TgAP-1-F and primer TgAP-1-R for detecting the SNP marker described in claim 1. The nucleotide sequence of the primer TgAP-1-F is shown in SEQ ID NO.

2. The nucleotide sequence of the primer TgAP-1-R is shown in SEQ ID NO.

3.

4. A kit for identifying arcobacter-resistant blood clams, characterized in that, The kit includes the primer set described in claim 3.

5. The kit according to claim 4, wherein The kit further includes a reagent for extracting genomic DNA from a sample and a reagent for performing a PCR reaction using the primer set described in claim 2.

6. Use of the SNP marker combination described in claim 1, or the primer set described in claim 2, or the kit described in any one of claims 4-5 in the identification of vibrio-resistant blood clams.

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