A molecular marker 10W6 for identifying WSSV resistance in Litopenaeus vannamei and its application

Abstract

The present invention provides a molecular marker 10W6 for identifying WSSV resistance of Penaeus vannamei and its application. The nucleotide sequence of the molecular marker 10W6 is shown in SEQ ID No.1, and the genotype CC of the 301st site of the molecular marker 10W6 is a WSSV resistance genotype. The present invention also provides primers for the molecular marker 10W6, including amplification primers with nucleotide sequences shown in SEQ ID No.2 and SEQ ID No.3 and an extension primer with a nucleotide sequence shown in SEQ ID No.4. The molecular marker 10W6 for high WSSV resistance of Penaeus vannamei is not limited by the growth stage, can be used for early breeding of Penaeus vannamei, screen WSSV high-resistant individuals in breeding populations with different growth performances, quickly breed parent populations with high WSSV resistance, and promote the breeding process of new varieties of Penaeus vannamei with high WSSV resistance.

Description

Technical Field

[0001] The invention belongs to the field of molecular marker-assisted breeding of aquatic animals, and specifically relates to a molecular marker 10W6 for identifying WSSV resistance of Litopenaeus vannamei and an application thereof. Background Art

[0002] Litopenaeus vannamei Penaeus vannamei ), also known as white shrimp, is native to the Pacific coast of South America. Due to its fast growth rate and strong environmental adaptability, it has been widely promoted for breeding since it was introduced to my country in 1988, and is a pillar industry of aquaculture in my country. Growth traits are the most concerned economic traits of Penaeus vannamei. However, with the continuous expansion of the scale of Penaeus vannamei breeding and the deterioration of the breeding environment, diseases frequently occur during the breeding process. Among them, the white spot syndrome virus (WSSV) has a strong transmission ability. The infected shrimps have reduced food intake, enlarged liver and pancreas, and white spots appear on the body. The mortality rate can reach 100% within 3-13 days, causing huge economic losses to the shrimp industry. At present, there is no effective method to prevent and control the spread of the virus. Cultivating new varieties with WSSV resistance is an urgent need of the market.

[0003] As of 2023, my country has bred 12 new varieties of Penaeus vannamei that have been approved by the state. The excellent traits of 11 varieties involve growth, and only the "Zhongxing No. 1" variety has shown resistance to the WSSV virus. The genetic correlation between growth and WSSV resistance ranges from moderate negative correlation to low positive correlation in different breeding populations of Penaeus vannamei, indicating that growth and WSSV resistance have different balance mechanisms in different breeding populations.

[0004] Single nucleotide polymorphism (SNP) markers have the characteristics of high richness, high density, strong stability, and co-dominance, and have become the most widely used molecular marker technology in economic crustaceans such as shrimp and crab. At present, there are few SNP markers related to WSSV resistance in Litopenaeus vannamei. The development of molecular markers that are significantly associated with WSSV resistance in different growth performance groups is of great significance for making full use of existing high-quality seed sources and accurately breeding new WSSV-resistant varieties. Summary of the invention

[0005] In view of the technical problems existing in the above-mentioned prior art, the purpose of the present invention is to provide a molecular marker 10W6 for identifying WSSV resistance of Penaeus vannamei and its application. The molecular marker 10W6 can be used in the screening of high white spot syndrome virus resistance of Penaeus vannamei, and has high identification efficiency and accuracy.

[0006] In order to solve the above technical problems, the technical solution to be adopted in the present invention is:

[0007] The present invention provides a molecular marker 10W6 for identifying WSSV resistance of Litopenaeus vannamei, characterized in that the nucleotide sequence of the molecular marker 10W6 is shown as SEQ ID No.1.

[0008] Furthermore, there is a C or A base mutation at the 301st base of the molecular marker 10W6.

[0009] Furthermore, the genotype CC at the 301st locus of the molecular marker 10W6 is a WSSV resistance genotype.

[0010] Further, the primers include amplification primers with nucleotide sequences as shown in SEQ ID No.2 and SEQ ID No.3 and extension primers with nucleotide sequences as shown in SEQ ID No.4.

[0011] The present invention provides the use of the molecular marker 10W6 or the primer of the molecular marker 10W6 in identifying a population of Penaeus vannamei with high WSSV resistance.

[0012] Furthermore, the specific steps for identifying a population of Penaeus vannamei with high WSSV resistance are as follows: extracting DNA of a sample to be tested in the Penaeus vannamei population, using it as a template, performing flight mass spectrometry typing using amplification primers and extension primers, and if the genotype of the 301st base in the typing result is CC, then the Penaeus vannamei population is selected as a parent for breeding Penaeus vannamei with high WSSV resistance.

[0013] Furthermore, the number of samples to be tested in the Penaeus vannamei population is more than 30.

[0014] Furthermore, in the flight mass spectrometry typing, the conditions for PCR amplification were pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at 56°C for 25 s, extension at 72°C for 30 s, repeated for a total of 40 cycles; final extension at 72°C for 3 min; and storage at 4°C.

[0015] Furthermore, in the flight mass spectrometry typing, the conditions of the extension reaction were pre-denaturation at 94°C for 30 s; denaturation at 94°C for 5 s, annealing at 56°C for 5 s, extension at 80°C for 5 s, repeated for 40 cycles; final extension at 72°C for 3 min; and storage at 4°C.

[0016] The invention provides the application of the molecular marker 10W6 in genetic diversity analysis, germplasm identification and genetic map construction of Penaeus vannamei.

[0017] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0018] 1. The high WSSV resistance molecular marker 10W6 of Penaeus vannamei provided by the present invention can be screened for WSSV high-resistant individuals in breeding populations with different growth performances without being restricted by growth performance, and a parent population with high WSSV resistance can be quickly bred, thereby promoting the breeding process of new varieties of Penaeus vannamei with high WSSV resistance.

[0019] 2. The molecular marker 10W6 provided by the present invention is used to detect the high WSSV resistance of Penaeus vannamei. The method is accurate, reliable, simple to operate, and can effectively and quickly screen out the high WSSV resistance population, increase the utilization rate of the existing germplasm of Penaeus vannamei, and improve the seed yield. It is of great significance to the healthy breeding and development of Penaeus vannamei and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the result of flight mass spectrometry typing validation in the validation population of the present invention. DETAILED DESCRIPTION

[0021] The present invention is further described in detail below in conjunction with embodiments. The following embodiments are for explanation of the present invention but the present invention is not limited to the following embodiments.

[0022] The vannamei shrimp used in the present invention are all from Bangpu Seed Technology Co., Ltd., a cooperative base of the Yellow Sea Fisheries Research Institute of the Chinese Academy of Fishery Sciences, and are commercially available shrimp. 59 full-sib families aged 77-94 days were selected, and all were negative for WSSV, EHP, AHPND, IHHNV, and DIV1 pathogens. 40 tails were randomly selected from each family, and a total of 2,360 tails were transported to the Genetic Breeding Center of the Yellow Sea Fisheries Research Institute (Jimo, Qingdao, Shandong) for growth and WSSV resistance tests.

[0023] Before the infection test, each family was placed in a 200 L plastic bucket for 4 days. During the period, the water temperature was controlled at 25 ± 1 ° C, 150 L of seawater was added, the salinity was 30, oxygen was continuously supplied, the bottom was sucked every day, and 50% of the water was changed. 12% of the body weight was weighed every day and fed three times at 8:00, 14:00, and 20:00.

[0024] The day before the infection test, feeding was stopped and the intestines were emptied. In the formal test, 30 shrimps were randomly selected from the temporary individuals of each family, totaling 1770 experimental group individuals. Referring to the method of feeding poison bait in the patent of Meng Xianhong et al. (patent number: ZL201210107377.8), 10 mg of poison bait was fed to each shrimp, and the virus content in the poison bait was 10 610 copies / mg. After feeding, each family was divided into three parallel groups, with 10 shrimps in each group, and placed in an acrylic box of 70 cm×40 cm×40 cm. The acrylic boxes were placed in three tanks with the same environmental conditions. During the breeding period, feed was fed and water was changed regularly every day to ensure the same breeding conditions. Five individuals from each family were taken as the control group and placed in another workshop. The breeding conditions were the same as those of the experimental group. No deaths occurred in the control group during the infection test. After the experimental group was fed with poison bait, it was observed every 2 hours, and the dead juvenile shrimps were fished out. The WSSV content in the tissue was detected using a probe quantitative kit to determine that the cause of death of the dead individuals was WSSV infection. The individual family number, individual death time and body length at the time of death (length from the end of the eyestalk to the end of the tail section) were recorded. After the peak of death, the experiment was stopped when there was no death for 3 consecutive days. Muscle samples were taken from all experimental groups, quickly frozen in liquid nitrogen, and stored in an ultra-low temperature refrigerator at -80℃.

[0025] The variance components and genetic parameters of body length and anti-WSSV survival time were estimated using the individual animal model, and the breeding values ​​of body length and anti-WSSV survival time of all individuals were estimated by the best linear unbiased prediction method. According to the growth breeding values, the fast-growing group and the slow-growing group were screened out; in the growth difference group, the high-resistance group and the low-resistance group were screened out according to the individual anti-WSSV survival time breeding values. In this way, four extreme groups were screened out, namely A: slow-growth and weak-resistance group, B: slow-growth and strong-resistance group, C: fast-growth and weak-resistance group, and D: fast-growth and strong-resistance group. Each group had 30 individuals, and the individuals between groups did not cross and had significant differences in the breeding values ​​of growth and WSSV survival time traits ( P <0.05). The body length and anti-WSSV survival time ranges of the four extreme mixed pool individuals are shown in Table 1.

[0026] Table 1: Body length and survival time range of WSSV in four extreme mixed pools

[0027]

[0028] Example 1

[0029] 1. Screening of candidate molecular markers associated with WSSV resistance

[0030] 1. High-throughput sequencing and sequence alignment

[0031] The CTAB (Cetyltrimethylammonium Bromide) method was used to extract DNA from muscle tissue of Penaeus vannamei tail by tail. Add 1 ml 1×CTAB to a 1.5 ml sterile enzyme centrifuge tube; take about 20 mg of sample and add it to the centrifuge tube, grind it at 60Hz for 4 minutes; incubate in a 65℃ water bath for 60 minutes; centrifuge at 8000g at room temperature for 5 minutes; take 900 μL of the supernatant of the sample after centrifugation to a new 2 mL sterile enzyme centrifuge tube; add 450 μL of chloroform to the supernatant; cover the tube tightly and mix the sample by inversion for 30 seconds until the solution is completely emulsified and turns white; centrifuge at 13000g at room temperature for 10 minutes; transfer 800 μL of the supernatant to a new 1.5 mL sterile enzyme-free centrifuge tube; Beckman AgencourtAMPure XP beads, balance at room temperature and away from light for 30 minutes; add 0.6 times the volume of the supernatant Beads, mix them evenly with a pipette for more than 10 times, and let them stand at room temperature for 5 minutes; place on a magnetic rack for 5 min, until the solution is clear, carefully aspirate and discard the supernatant; keep the above 1.5 mL centrifuge tube fixed on the magnetic rack, add 200 μL of freshly prepared 80% ethanol to the tube, let it stand at room temperature for 30 s, and discard the supernatant, taking care not to disturb the magnetic beads; repeat the 80% ethanol wash once; keep the 1.5 mL centrifuge tube fixed on the magnetic rack, and dry the magnetic beads at room temperature for 2~5 min; remove the 1.5 mL centrifuge tube from the magnetic rack, add 50 μL of 10 mM Tris HCl elution solution, gently blow and mix with a pipette, and let it stand at room temperature for 5 min; place the 1.5 mL centrifuge tube on the magnetic rack, let it stand at room temperature for 5 min until the solution is clear, carefully aspirate about 50 μL of the supernatant and transfer it to the corresponding new sample storage tube to obtain the purified DNA. The purity and integrity of the DNA were analyzed by agarose gel electrophoresis; the purity of the DNA (OD260 / 280 ratio) was detected by Nanodrop, and the DNA concentration was accurately quantified by Qubit.

[0032] Each pool of 30 individuals provided an equal amount of DNA samples, and four DNA pools were constructed, namely A with slow growth and weak resistance, B with slow growth and strong resistance, C with fast growth and weak resistance, and D with fast growth and strong resistance. The mixed DNA samples were randomly broken into fragments of 500 bp in length by CovarisS2 / E210 crusher, and the DNA fragments were prepared by end repair, ployA tail addition, sequencing adapter addition, purification, PCR amplification and other steps to complete the entire library preparation. The constructed library was sequenced by Illumina Novaseq 6000 sequencing platform, and the sequencing data was quality controlled to finally obtain CleanData data, which was compared with the reference genome to complete the detection and annotation of the site. The quality statistics of the sequencing data of the four pools are shown in Table 2. The filtered valid data were aligned by Burrows-Wheeleralignment tool (BWA) software, and the alignment results were removed by SAMTOOLS software to remove PCR duplication. The average sequencing depth was above 30×, which can be used for subsequent analysis.

[0033] Table 2: Overview of sequencing quality control

[0034]

[0035] 2. Screening of genomic differential regions

[0036] Different genomic regions were selected based on the sliding window strategy: 50 kb as the unit window and 25 kb as the sliding step. The nucleotide diversity difference (π-Ratio) and genetic differentiation coefficient (Fixation index, Fst) between the AB group and the CD group were calculated for the selected specific genomic regions. Combining the results of π-Ratio and Fst, each was calculated according to the difference between the populations. P <0.05 was used to select significant regions.

[0037] 3. Marker Detection and Annotation

[0038] The Unified Genotyper module in the Genome analysis toolkit (GATK) software was used to detect SNPs and InDels in the candidate intervals, and the filtering parameters were set as follows: -Window 4, -filter "QD < 2.0 || FS > 60.0 || MQ < 40.0 ", -G_filter "GQ < 20". 697 selected genomic regions were located between the AB group, containing 510,524 SNP sites; 842 selected genomic regions were located between the CD group, containing 585,675 SNP sites.

[0039] 4. SNP frequency difference analysis

[0040] The SNP-index of each site in the AB population and the CD population pool was calculated separately, and the frequency difference distribution of SNP was calculated at the same time, and the direction was as follows: △(index) =index (high WSSV resistance) - index (low WSSV resistance).

[0041] 5. Marker screening

[0042] The candidate SNPs were screened and the sites with △index close to 1 or close to -1 in the two populations were selected as the priority sites for the next step of verification. The screening criteria are as follows:

[0043] (1) Based on the annotation information of the SNP loci, and then sorted from high to low according to │△index│, the sites of synonymous, non-synonymous mutations or upstream and downstream regions are given priority;

[0044] Finally, 69 markers with large frequency differences between the AB and CD groups were screened out.

[0045] Table 3: Statistical results of SNPs detection and annotation among resistance difference groups

[0046]

[0047] 2. Verification of molecular markers associated with WSSV resistance

[0048] Time-of-flight mass spectrometry was used to verify candidate molecular markers associated with WSSV resistance in the slow-growing and weak-resistance, slow-growing and strong-resistance, fast-growing and weak-resistance, and fast-growing and strong-resistance groups, with 30 individuals in each group. The specific steps are as follows:

[0049] (1) PCR amplification: Design primers on the flanking sequence of the marker site to amplify the DNA sequence containing the detection target;

[0050] (2) Single base extension: SNP sequence-specific extension primers are added to the PCR amplification product, and single base extension is performed using iPLEX technology. For the detection target, different genotypes only differ by one base at the end of the extension;

[0051] (3) Mass spectrometry detection: The product after single-base extension is purified and transferred to the SpectroCHIP chip for mass spectrometry detection. DNA is positively charged by laser irradiation and flies in the detection vacuum tube. The flying speed is inversely proportional to the mass of each extension product. Finally, the base type is determined by the position of the peak, thereby achieving genotyping;

[0052] (4) The mutation markers of each individual were counted based on the mass spectrometry detection results, and the correlation between the markers and WSSV resistance was analyzed using SPSS software.

[0053] The specific steps are as follows:

[0054] 1. Primer design

[0055] Primer design software Sequenom's Genotyping Tools and Massarray Assay Design software were used to design PCR amplification primers and single-base extension primers for the loci. Primer length was 18-30 bp, PCR product 80-200 bp), T m Value 55-65℃, annealing temperature around 60℃; GC content 40-70%.

[0056] 2. PCR amplification

[0057] The total volume of each amplification reaction system was 5 μL. The PCR amplification reaction system was shown in Table 4, and the reaction procedure was shown in Table 5. Primer synthesis and sequencing were completed by Shanghai Sangon Biotechnology Co., Ltd.

[0058] Table 4: PCR amplification reaction system components

[0059]

[0060] Table 5: PCR amplification reaction program

[0061]

[0062] 3. Treatment of PCR products with alkaline phosphatase

[0063] (1) After the PCR reaction is completed, SAP (shrimp alikaiine phosphtase) is used to purify the multiple PCR amplification reaction products to remove dNTPs in the reaction. The SAP reaction solution formula is as follows (taking a single sample as an example): SAP Buffer 0.17 μl, SAP Enzyme (1.7U / μl) 0.3 μl, ddH2O 1.53 μl;

[0064] (2) Use a pipette to add the SAP reaction solution to the PCR reaction plate, add 2 µl to each well, seal the plate, and centrifuge;

[0065] (3) Place the PCR reaction plate with SAP reaction solution in a PCR instrument and run the following reaction program: 37°C, 40 min; 85°C, 5 min; store at 4°C;

[0066] (4) After the reaction is completed, remove the PCR reaction plate and centrifuge it briefly for later use.

[0067] 4. Single base extension reaction

[0068] The addition of ddSNP allowed the PCR to extend only one base. The single base extension reaction system was shown in Table 6, and the total volume of the reaction system was 2 μL. The reaction procedure was shown in Table 7.

[0069] Table 6: Single base extension reaction system

[0070]

[0071] Table 7: Single base extension reaction program

[0072]

[0073] 5. Product purification

[0074] (1) Evenly cover the resin on the resin scraper and leave it for 20 minutes;

[0075] (2) Centrifuge the PCR reaction plate at 1000 rpm for 1 min, add 25 µl of deionized water to each well, invert it on the resin plate (make sure it is fixed and cannot be moved), then invert the resin plate and put it on the PCR reaction plate, tap it to make the resin fall into the PCR reaction plate, and seal it with film;

[0076] (3) With the long axis of the PCR reaction plate as the axis, turn the PCR reaction plate over for 20 minutes. Centrifuge at 3500 rpm for 5 minutes and set aside.

[0077] 6. Mass spectrometry

[0078] (1) Nanodispenser SpectroCHIP chip spotting transfers the test sample from the PCR reaction plate to the MassARRAY SpectroCHIP chip with a surface covered with matrix;

[0079] (2) MassARRAY Analyzer Compac mass spectrometry detection;

[0080] (3) TYPER software was used to analyze the experimental results and obtain typing data.

[0081] 7. Statistical analysis

[0082] As shown in Table 8, the proportion of genotype CC in the slow growth and strong resistance group and the fast growth and strong resistance group in 10W6 was 0.63 and 0.53 respectively, both of which were dominant genotypes. P=0.001, which is extremely significant, so the genotype CC at this site can be considered to be a WSSV resistance genotype. The nucleotide sequence of the 10W6 molecular marker is shown in SEQ ID No.1, wherein the amplification primers for developing the molecular marker are shown in SEQ ID No.2 and SEQ ID No.3, and the extension primer is shown in SEQ ID No.4 (Table 9).

[0083] Table 8: Typing results of 10W6

[0084]

[0085] Table 9 Primers for molecular markers

[0086]

[0087] The molecular marker 10W6 obtained by the present invention can be used to assist in selecting a high WSSV-resistant variety of Penaeus vannamei, and the application steps are specifically as follows: extracting DNA of a test sample of Penaeus vannamei and using it as a template, using amplification primers 10W6-F and 10W6-R of the molecular marker 10W6 and an extension primer 10W6-E to perform flight mass spectrometry typing, and if the 301st base genotype of 10W6 in the typing result is CC, the test sample can be selected as a parent for breeding a high WSSV-resistant variety of Penaeus vannamei.

[0088] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to replace some of the technical features therein by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions claimed to be protected by the present invention.

Claims

1. A molecular marker 10W6 for identifying WSSV resistance in Penaeus vannamei, characterized in that: The nucleotide sequence of the molecular marker 10W6 is shown in SEQ ID No. 1; there is a C or A base mutation at the 301st base of the molecular marker 10W6, and the genotype CC at the 301st site of the molecular marker 10W6 is a WSSV resistance genotype.

2. The primer of the molecular marker 10W6 according to claim 1, characterized in that The primers include amplification primers whose nucleotide sequences are shown as SEQ ID No.2 and SEQ ID No.3 and extension primers whose nucleotide sequences are shown as SEQ ID No.

4.

3. Use of the molecular marker 10W6 according to claim 1 or the primer of the molecular marker 10W6 according to claim 2 in identifying a population of Penaeus vannamei with high WSSV resistance.

4. The use according to claim 3, characterized in that: The specific steps of identifying a population of Penaeus vannamei with high WSSV resistance are: extracting DNA of a sample to be tested in the Penaeus vannamei population, using the DNA as a template, performing flight mass spectrometry typing using amplification primers with nucleotide sequences as shown in SEQ ID No. 2 and SEQ ID No. 3 and an extension primer with a nucleotide sequence as shown in SEQ ID No. 4, and if the genotype of the 301st base is CC in the typing result, the Penaeus vannamei population is selected as a parent for breeding Penaeus vannamei with high WSSV resistance.

5. The use according to claim 4, characterized in that: The number of samples to be tested in the vannamei shrimp population is more than 30.

6. The use according to claim 4, characterized in that: In the flight mass spectrometry typing, the conditions for PCR amplification were pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at 56°C for 25 s, extension at 72°C for 30 s, repeated for a total of 40 cycles; final extension at 72°C for 3 min; and storage at 4°C.

7. The use according to claim 4, characterized in that: In the flight mass spectrometry typing, the conditions of the extension reaction were pre-denaturation at 94°C for 30 s; denaturation at 94°C for 5 s, annealing at 56°C for 5 s, extension at 80°C for 5 s, repeated for 40 cycles; final extension at 72°C for 3 min; and storage at 4°C.

8. Use of the molecular marker 10W6 according to claim 1 in genetic diversity analysis, germplasm identification and genetic map construction of Penaeus vannamei.

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