SNP molecular marker related to WSSV tolerance character of procambarus clarkii and application thereof

Abstract

The invention discloses an SNP (Single Nucleotide Polymorphism) molecular marker related to the tolerance character of white spot syndrome virus (WSSV) of procambarus clarkia, the molecular marker is located in the third intron region of a TRIM gene, is specifically located at 585bp of a DNA (Deoxyribose Nucleic Acid) sequence as shown in SEQ ID NO.1, and is Tgt; the T / C heterozygous genotype is obviously related to the WSSV tolerance character of the procambarus clarkia. The invention provides a favorable marker for breeding of disease-resistant molecules of procambarus clarkii, and can accelerate the breeding process of disease-resistant varieties of procambarus clarkii.

Description

Technical Field

[0001] The invention relates to a SNP molecular marker related to the tolerance trait of white spot syndrome virus (WSSV) of Procambarus clarkii, and belongs to the field of molecular biology. Technical Background

[0002] Procambarus clarkii, commonly known as "crayfish", belongs to the phylum Arthropoda, class Crustacea, phylum Decapoda, family Crayfish, genus Procambarus, native to the United States and Mexico, and is one of the most popular aquatic products in my country. Procambarus clarkii has a unique taste, high muscle protein content and rich nutrition. According to the "2024 China Crayfish Industry Development Report", the production of crayfish in China reached 3.161 million tons in 2023, and the total breeding area reached 29.5 million mu.

[0003] WSSV is a double-stranded DNA virus that can infect almost all crustaceans such as shrimp, lobster, crayfish, crab, copepods, etc. It has the characteristics of strong transmission ability and high mortality rate (Lei Zhiwen et al. 2002). Once shrimp is infected with WSSV, the mortality rate is as high as 100% within 3-10 days (Yan Dongchun 2006). WSSV is the most threatening viral pathogen of Procambarus clarkii. Studies have shown that Procambarus clarkii is infected with WSSV throughout the year, with the highest virus infection rate in April and May, reaching 81.12% and 88.83% (Luo Xiaohong 2023), which is one of the important factors affecting the healthy development of Procambarus clarkii aquaculture.

[0004] Most of the tripartite motif protein (TRIM) family contains a RING domain with a zinc finger motif and has E3 ubiquitin ligase activity (Joazeiro and Weissman, 2000). They play a key role in biological processes such as innate immunity, anti-tumor immunity, oxidative stress, and cell cycle regulation. TRIM proteins protect hosts from viral infection in a variety of ways. On the one hand, TRIM proteins are believed to be involved in inhibiting the replication of a variety of viral nucleic acids (Yang et al. 2016; Full et al. 2019). On the other hand, TRIM proteins are involved in the regulation of cellular autophagy and can block the escape of viruses from host cells by inhibiting phagocytosis (Sartute et al. 2019). In crustaceans, studies have shown that TRIM proteins located on the cell membrane can recognize WSSV and interact with Activating protein 1 (AP1), inhibiting the expression of endocytosis-related dynamin, reducing the endocytosis of WSSV by cells, and inhibiting viral infection at the source (Cao Xiaotong 2023).

[0005] Single nucleotide polymorphism (SNP) is a third-generation molecular marker that is widely present in various locations of the biological genome and has the characteristics of high genetic stability. With the development of high-throughput typing technology, SNP markers have been widely used in aquatic animal breeding (Yu Yang 2014; Zhao Ji 2021; Liu Yang et al. 2023). At present, the difficulty in defining disease resistance traits and the inability to stably inherit disease resistance are common problems in disease-resistant breeding. Molecular markers based on functional genes can shorten the breeding cycle and improve breeding efficiency (Ribaut and Hoisington 1998). Therefore, the development of SNP markers related to WSSV tolerance traits can provide a basis for the selection and breeding of Procambarus clarkii varieties. Summary of the invention

[0006] The purpose of the present invention is to provide a SNP molecular marker related to the WSSV tolerance trait of Procambarus clarkii for use in the molecular breeding of disease resistance of Procambarus clarkii.

[0007] To achieve the above objectives, the applicant conducted BSA resequencing analysis on crayfish populations with different virus copy numbers and found a SNP molecular marker that was significantly associated with the WSSV tolerance trait of crayfish. The polymorphic site of the SNP molecular marker is located in the third intron region of the TRIM gene of crayfish, and the TRIM gene has been shown to be involved in the early anti-WSSV immune response of crayfish.

[0008] Furthermore, the crayfish was challenged with WSSV, and PCR primers were designed for amplification and sequencing after DNA extraction. The chi-square test was used to test the significant difference between genotype and WSSV tolerance trait. Finally, it was verified that a SNP site in the third intron region of the TRIM gene was significantly correlated with the WSSV tolerance trait of crayfish, which can be used for the identification of WSSV tolerance traits and molecular breeding of crayfish.

[0009] Specifically, the SNP molecular marker is located at the 585bp of the DNA sequence shown in SEQ ID NO.1, and the base is T or C, wherein the T / C heterozygous genotype is a favorable marker for the WSSV tolerance trait of Procambarus clarkii, and the DNA sequence is a fragment of the third intron region of the TRIM gene of Procambarus clarkii.

[0010] SEQ ID NO.1:

[0011]

[0012] The bold sequence is the SNP site; the sequence shown by the wavy line is the primer for amplifying the molecular marker.

[0013] The present invention also provides a method for identifying the WSSV tolerance trait of Procambarus clarkii, the method comprising the following steps:

[0014] 1) Extracting genomic DNA of Procambarus clarkii;

[0015] 2) Using the extracted genomic DNA as a template, designing PCR primers to amplify a fragment containing the SNP molecular marker;

[0016] 3) Sequencing the PCR amplification product to detect the genotype of the SNP molecular marker.

[0017] The sequences of the PCR primers are shown in SEQ ID NO.3 and SEQ ID NO.4, which are respectively the upstream primer and the downstream primer for amplifying the DNA sequence shown in SEQ ID NO.1.

[0018] The PCR amplification system is as follows: 25 μL of 2×Es Taq MasterMix (Dye), 2 μL of 10 nM upstream primer and downstream primer, 1 μL of 100 ng / L DNA template, and ddH2O to make up the volume to 50 μL.

[0019] The PCR amplification procedure is as follows: pre-denaturation at 94°C for 2 min, each cycle includes denaturation at 94°C for 30 s, annealing at 56°C for 30 s, and extension at 72°C for 30 s, for a total of 35 cycles; and extension at 72°C for 2 min after the cycle ends.

[0020] Among them, if the test result is T / C heterozygous genotype, the crayfish is WSSV-tolerant.

[0021] The beneficial effects of the present invention are:

[0022] The present invention provides a new SNP molecular marker associated with the WSSV tolerance trait of Procambarus clarkii, which can be used to quickly and accurately identify Procambarus clarkii tolerant to WSSV, thereby providing favorable technical support for the disease-resistant breeding of Procambarus clarkii and accelerating the variety breeding process. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the distribution diagram of white spot syndrome virus load in 313 Procambarus clarkii crayfish.

[0024] Figure 2 Schematic diagram of the TRIM gene structure.

[0025] Figure 3 This is the sequencing peak diagram of different genotypes at the 585bp position of the nucleotide sequence shown in SEQ ID NO.1. DETAILED DESCRIPTION

[0026] The present invention is further described below in conjunction with specific embodiments.

[0027] Example 1: Acquisition of SNPs associated with WSSV tolerance traits

[0028] 1. Construction of sequencing population

[0029] 313 samples of gill and muscle tissues of Procambarus clarkii from the same breeding pond were randomly collected and quickly frozen in liquid nitrogen for genomic DNA extraction. The number of WSSV copies in the gill tissue of each shrimp was detected by real-time fluorescence qPCR using primers WDP07: 5'-ACAACTAGCAAGCACTGGTAT-3' and WDP06: 5'-TCAGCTTCAAGAATCCCAATAG-3'. The results are shown in Figure 2. Figure 1 Then, 15 individuals with extremely low virus copy numbers and 15 individuals with extremely high virus copy numbers were selected to form two gene pools. The virus copy numbers of these two pools are shown in Table 1. Table 1 shows the sequencing population of the mixed pool of Procambarus clarkii. The 1st to 15th tails belong to the group with extremely low virus copy numbers, and the 16th to 30th tails belong to the group with extremely high virus copy numbers.

[0030] Table 1 Virus carrying status of mixed pool sequencing population

[0031]

[0032]

[0033] 2. BSA resequencing analysis

[0034] DNA was extracted from muscle tissue of the above two groups, and high and low viral copy number pools were constructed by mixing equal amounts of DNA. After the DNA sample was qualified, the genomic DNA was fragmented by ultrasound to construct a small DNA fragment library, and the high and low viral copy number pools were sequenced by Illumina Hiseq 2500 sequencer with high-throughput sequencing, and the sequencing depth was 30X. The mutation analysis software GATK was used to detect the SNP method of the population, and further filtering and screening were performed according to factors such as quality value, depth, and repeatability. Finally, high-confidence variant sites were obtained, and the detected candidate SNPs were annotated accordingly using ANNOVAR software and the published genome annotation file (GCF_040958095.1). The candidate regions of the research target traits were obtained by screening the SNP-index difference regions, and the initial positioning of the target SNP was completed.

[0035] A total of 16,943,166 SNP sites were detected in this BSA resequencing, and 4,415,692 SNP sites were obtained after filtering. After comparison, it was found that there were 2 fragments on the TRIM gene that may contain SNP sites related to the WSSV tolerance trait.

[0036] 3. Bioinformatics analysis of TRIM gene cDNA sequence of Procambarus clarkii

[0037] According to the full-length cDNA sequence information of TRIM gene of Procambarus clarkii reported in Genbank (XP_045581296), its amino acid coding sequence was searched by Blast, and the distribution of introns, exons and 5'-flanking regulatory region of TRIM gene of Procambarus clarkii was predicted by comparing the Blast results.

[0038] The TRIM gene of Procambarus clarkii is 12691 bp in length, including 4 exons and 3 introns. Among them, extron1 is 212 bp, extron2 is 218 bp, extron3 is 100 bp, and extron4 is 7077 bp. Intron1 is 1989 bp, intron2 is 239 bp, intron3 is 2856 bp, and the 5'-flanking regulatory region sequence is 1000 bp. The sequence structure information is shown in Figure 2 .

[0039] Example 2: Screening of TRIM gene SNPs in Procambarus clarkii and analysis of their correlation with WSSV tolerance traits

[0040] 1. Test methods

[0041] 1.1 Separation of sensitive and tolerant populations

[0042] The crayfish samples needed for this experiment were from the Crayfish Breeding Base of Huazhong Agricultural University (Caidian). They were kept in polyethylene plastic culture boxes for 3 days before the experiment, and 300 healthy and vigorous crayfish were selected for the experiment. The indoor culture water temperature was 24-25℃. During the experiment, commercial feed was fed once a day, and the water was changed once every two days. 300 healthy crayfish were injected with 0.1mL WSSV (10 5 The injection site was the second abdominal segment. As the virus replicated and spread in the shrimp, the first 50 crayfish that died of the disease were used as the sensitive group. After 15 days, the 50 surviving crayfish were used as the tolerant group.

[0043] 1.2 Extraction of genomic DNA

[0044] The tail muscles of two groups of Procambarus clarkii were collected and stored in 2 mL centrifuge tubes filled with anhydrous ethanol. DNA was extracted according to the instructions of the kit and the obtained DNA samples were stored at -20°C.

[0045] 1.3 Primer design

[0046] According to the obtained DNA sequence of TRIM gene of Procambarus clarkii (SEQ ID NO2), primers were designed using Primer 6.0 software to amplify the potential SNP site fragment. The primer sequences are shown in Table 2.

[0047] Table 2 SNP primer sequences designed for two fragments on the TRIM gene

[0048]

[0049] 1.4 PCR amplification

[0050] The DNA of Procambarus clarkii in the tolerant group and the sensitive group was used as template, and PCR amplification was performed with the primers shown in Table 2. The amplification system (Kangwei Century: 2×Es Taq MasterMix (Dye) 25μL, upstream primer and downstream primer 2μL (10nM) each, DNA template 1μL (100ng / L), ddH2O made up the volume to 50μL. The PCR reaction was performed for a total of 35 cycles, with pre-denaturation at 94℃ for 2min, and each cycle included denaturation at 94℃ for 30s, annealing at 56℃ for 30s, and extension at 72℃ for 30s; after the cycle, extension was performed at 72℃ for 2min.

[0051] 1.5 Small-scale population SNP screening and large-scale population SNP verification

[0052] 20 individuals were randomly selected from the tolerant group and the sensitive group, and the designed primers were used to perform PCR amplification on the DNA of the tolerant group and the sensitive group. 2 μL of the amplified product was detected by 1.5% agarose gel electrophoresis, and the PCR product with clear and bright target band was selected and sent to Wuhan Hece Gene Technology Co., Ltd. for sequencing. The sequencing results were analyzed by SnapGene software for nucleotide sequence analysis and sequencing peak comparison. The sites with significant differences in the tolerant group and the sensitive group in a small population were verified by single individuals in the remaining 60 individuals.

[0053] 1.6 Data Analysis

[0054] The genotype frequencies of each SNP on TRIM gene fragment 1 and fragment 2 were statistically calculated, and the chi-square test between each SNP genotype and tolerance trait was performed using SPSS22.0.

[0055] 2. Results Analysis

[0056] 2.1 Small-scale population screening of TRIM gene SNPs in Procambarus clarkii

[0057] The TRIM gene fragments 1 and 2 of Procambarus clarkii were amplified and sequenced. After comparison, 9 SNP sites were found in fragments 1 and 2. In fragment 2, a SNP site with significant differences between the tolerant group and the sensitive group was found. After comparison, this site was located in the third intron region of the TRIM gene, as shown in Table 3.

[0058] Table 3. Correlation analysis between TRIM gene SNPs and WSSV tolerance traits in Procambarus clarkii

[0059]

[0060]

[0061]

[0062] Note: Bold numbers indicate significant differences

[0063] 2.2 Association analysis between TRIM gene SNP loci and WSSV tolerance in Procambarus clarkii

[0064] The genotype frequencies of the five SNP sites on segment 2 in the tolerant group and the sensitive group were statistically analyzed, and the chi-square test in SPSS22.0 software was used to test the significant difference in the genotype frequencies of these five SNP sites in the tolerant group and the sensitive group. P<0.05 was considered to be a significant difference, otherwise there was no significant difference.

[0065] Table 4. Association analysis between TRIM gene SNPs and WSSV tolerance traits in Procambarus clarkii

[0066]

[0067]

[0068] Note: Bold numbers indicate significant differences

[0069] Table 4 shows that in g.3304T>C, the proportion of TC genotype crayfish in the tolerant group was higher than that in the sensitive group. The chi-square test showed that the frequency of g.3304T>C genotype was significantly different in both the tolerant group and the sensitive group (P<0.05). These results indicate that g.3304T>C is significantly correlated with WSSV tolerance, and g.3304T>C is the dominant genotype in the WSSV tolerance group. Therefore, WSSV tolerance breeding can be carried out by selecting crayfish with TC genotype at the TRIM gene g.3304T>C locus.

[0070] References

[0071] 1. Lei Zhiwen, Huang Jie, Liang Chengzhu, et al. Biological characteristics of white spot syndrome virus[J]. Marine Science, 2002, 26(3): 26-31.

[0072] 2. Yan Dongchun. Main measures for the prevention and control of white spot syndrome virus (WSSV) in shrimp[J]. Fisheries Science, 2006, 25(4): 202-204.

[0073] 3. Luo Xiaohong. Seasonal dynamics of white spot syndrome virus (WSSV) and microbial communities in Procambarus clarkii under different culture modes[D]. Hubei: Huazhong Agricultural University, 2023.

[0074] 4. Cao Xiaotong. Functional study of PcTRIM and ATF4-β in the process of Procambarus clarkii resisting white spot syndrome virus infection. Shandong Agricultural University, 2023.

[0075] 5. Yu Yang. Development of molecular markers for Penaeus vannamei and its application in genetic breeding[D]. University of Chinese Academy of Sciences, 2014.

[0076] 6. Zhao Ji. Genome-wide selection breeding of large yellow croaker (Larimichthys crocea) for resistance to Cryptocaryon irritans[D]. Fujian: Xiamen University, 2021.

[0077] 7. Liu Yang, Luan Sheng, Liu Mianyu, et al. Analysis of the accuracy of genomic prediction of AHPND resistance in Litopenaeus vannamei based on SNP panels of different densities[J]. Journal of Fisheries of China, 2023, 47(1): 163-172.

[0078] 8. Joazeiro CA, Weissman AM. RING finger proteins: mediators of ubiquitinligase activity. Cell. 2000; 102(5):549-552.

[0080] 9.Yang, C., Zhao,

[0081] 10.Full,F.,van Gent,M.,Sparrer,K.M.J.et al.Centrosomal protein TRIM43restrictsherpesvirus infection by regulating nuclear lamina integrity.NatMicrobiol.,(2019),4(1):164–176

[0083] 11.Sarute N,Ibrahim N,Medegan Fagla B,et al.TRIM2,a novel member ofthe antiviralfamily,limits New World arenavirus entry.PLoS Biol.2019;17(2):e3000137.

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Claims

1. A SNP molecular marker associated with the white spot syndrome virus (WSSV) tolerance trait of Procambarus clarkii, wherein the SNP molecular marker is located at the 585th bp of the DNA sequence shown in SEQ ID NO.1, the base is T or C, wherein the T / C heterozygous genotype is a favorable marker for the WSSV tolerance trait of Procambarus clarkii, and the DNA sequence is a fragment of the third intron region of the TRIM gene of Procambarus clarkii.

2. Use of the SNP molecular marker according to claim 1 in identifying the WSSV tolerance trait of Procambarus clarkii.

3. A method for identifying WSSV tolerance traits in Procambarus clarkii, characterized in that The following steps are involved: 1) Extracting genomic DNA of Procambarus clarkii; 2) using the extracted genomic DNA as a template, designing PCR primers to amplify a fragment containing the SNP molecular marker of claim 1; 3) Sequencing the PCR amplification product to detect the genotype of the SNP molecular marker according to claim 1.

4. The method for identifying WSSV tolerance of Procambarus clarkii according to claim 3, characterized in that: The sequences of the PCR primers are shown in SEQ ID NO.3 and SEQ ID NO.4, which are respectively the upstream primer and the downstream primer for amplifying the DNA sequence shown in SEQ ID NO.

1.

5. The method for identifying WSSV tolerance of Procambarus clarkii according to claim 4, characterized in that: The PCR amplification system is: 25 μL of 2×Es Taq MasterMix (Dye), 2 μL of 10 nM upstream primer and downstream primer respectively, 1 μL of 100 ng / L DNA template, and ddH2O to make up the volume to 50 μL.

6. The method for identifying WSSV tolerance of Procambarus clarkii according to claim 4, characterized in that: The PCR amplification procedure was as follows: pre-denaturation at 94°C for 2 min, each cycle including denaturation at 94°C for 30 s, annealing at 56°C for 30 s, and extension at 72°C for 30 s, for a total of 35 cycles; and extension at 72°C for 2 min after the cycle was completed.

7. The method for identifying WSSV tolerance of Procambarus clarkii according to claim 3, characterized in that: If the test result is a T / C heterozygous genotype, the crayfish is WSSV-tolerant.

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