A SNP molecular marker related to wssv tolerance trait of procambarus clarkii and application thereof

Abstract

The application discloses a SNP molecular marker related to a white spot syndrome virus (WSSV) tolerance trait of Procambarus clarkii, which is located in a 3rd intron region of a TRIM gene, specifically, a T>C type mutation at 585bp of a DNA sequence shown in SEQ ID NO. 1, wherein a T / C heterozygous genotype has significant correlation with the WSSV tolerance trait of the Procambarus clarkii. The application provides a favorable marker for molecular selection of the Procambarus clarkii, and can accelerate the selection process of the disease-resistant variety of the Procambarus clarkii.

Description

Technical Field

[0001] This invention relates to a SNP molecular marker associated with tolerance to white spot syndrome virus (WSSV) in red swamp crayfish, belonging to the field of molecular biology. Technical Background

[0002] The red swamp crayfish, commonly known as "crayfish," belongs to the phylum Arthropoda, class Crustacea, phylum Decapoda, family Palaemonidae, and genus Procambarus. Originally from the United States and Mexico, it is one of the most popular aquatic products in my country. The red swamp crayfish has a unique flavor, high muscle protein content, and is rich in nutrients. According to the "2024 China Crayfish Industry Development Report," in 2023, China's crayfish production reached 3.161 million tons, with a total farming area of ​​29.5 million mu (approximately 1.67 million hectares).

[0003] WSSV is a double-stranded DNA virus that can infect almost all crustaceans, including shrimp, lobsters, crayfish, crabs, and copepods. It is characterized by its high transmissibility and mortality rate (Lei et al., 2002). Once shrimp are infected with WSSV, the mortality rate can reach 100% within 3-10 days (Yan Dongchun, 2006). WSSV is the most threatening viral pathogen in the red swamp crayfish (Procambarus clarkii). Studies have shown that WSSV infection in red swamp crayfish occurs year-round, with the highest infection rates in April and May, reaching 81.12% and 88.83% respectively (Luo Xiaohong, 2023). It is one of the important factors affecting the healthy development of red swamp crayfish aquaculture.

[0004] Most tripartite motif proteins (TRIMs) contain a RING domain with a zinc finger motif and possess E3 ubiquitin ligase activity (Joazeiro and Weissman, 2000). They play crucial roles in biological processes such as innate immunity, anti-tumor immunity, oxidative stress, and cell cycle regulation. TRIM proteins protect the host from viral infection in multiple ways. On the one hand, TRIM proteins are thought to participate in inhibiting the replication of various viral nucleic acids (Yang et al. 2016; Full et al. 2019). On the other hand, TRIM proteins participate in the regulation of autophagy and can block viral escape 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 dynamin, an endocytosis-related dynamogen, thereby reducing cellular endocytosis of WSSV and inhibiting viral infection at its source (Cao Xiaotong 2023).

[0005] Single nucleotide polymorphisms (SNPs) are third-generation molecular markers, widely distributed across various locations in the genome of organisms, and characterized by high genetic stability. With the development of high-throughput genotyping technology, SNP markers have been widely used in aquatic animal breeding (Yu Yang 2014; Zhao Ji 2021; Liu Yang et al. 2023). Currently, the difficulty in defining disease resistance traits and the inability to stably inherit disease resistance are common problems in disease resistance breeding. Molecular markers based on functional genes can shorten the breeding cycle and improve breeding efficiency (Ribaut and Hoisington 1998). Therefore, developing SNP markers related to WSSV tolerance traits can provide a basis for the breeding of red swamp crayfish varieties. Summary of the Invention

[0006] The purpose of this invention is to provide an SNP molecular marker associated with the WSSV tolerance trait of Procambarus clarkii for the selection of disease-resistant molecules in Procambarus clarkii.

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

[0008] Furthermore, WSSV was challenged in *Procambarus clarkii*, and DNA was extracted. PCR primers were designed for amplification and sequencing. Chi-square test was used to examine the significant difference between genotype and WSSV tolerance traits. Ultimately, it was verified that a SNP site in the third intron region of the TRIM gene was significantly associated with WSSV tolerance traits in *Procambarus clarkii*, which can be used for the identification and molecular selection of WSSV tolerance traits in *Procambarus clarkii*.

[0009] Specifically, the SNP molecular marker is located at the 585th bp of the DNA sequence shown in SEQ ID NO.1, and the base is T or C. 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 bolded sequences are SNP sites; the wavy lines indicate the primers used to amplify the molecular marker.

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

[0014] 1) Extract genomic DNA from the red swamp crayfish;

[0015] 2) Using the extracted genomic DNA as a template, design PCR primers to amplify fragments containing the SNP molecular markers;

[0016] 3) Sequencing the PCR amplification products 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 the upstream and downstream primers for amplifying the DNA sequence shown in SEQ ID NO.1, respectively.

[0018] The PCR amplification system consisted of: 25 μL of 2×Es Taq MasterMix (Dye), 2 μL each of 10 nM upstream and downstream primers, 1 μL of 100 ng / L DNA template, and ddH2O to bring the volume to 50 μL.

[0019] The PCR amplification program is as follows: pre-denaturation at 94℃ for 2 min, each cycle consisting of denaturation at 94℃ for 30 s, annealing at 56℃ for 30 s, extension at 72℃ for 30 s, for a total of 35 cycles; after each cycle, extension at 72℃ for 2 min.

[0020] If the test result is a T / C heterozygous genotype, then the red swamp crayfish exhibits WSSV tolerance.

[0021] The beneficial effects of this invention are:

[0022] This invention provides a novel SNP molecular marker associated with WSSV tolerance in Procambarus clarkii. This marker can be used to quickly and accurately identify WSSV-tolerant Procambarus clarkii, thereby providing favorable technical support for disease-resistant breeding of Procambarus clarkii and accelerating the breeding process. Attached Figure Description

[0023] Figure 1 The distribution map of white spot syndrome virus load in 313 red swamp crayfish.

[0024] Figure 2 This is a schematic diagram of the TRIM gene structure.

[0025] Figure 3 The sequence diagram shows the sequencing peaks of different genotypes at the 585bp position of the nucleotide sequence shown in SEQ ID NO.1. Detailed Implementation

[0026] The present invention will be further described below with reference to specific embodiments.

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

[0028] 1. Construction of the sequencing population

[0029] Thirty-three gill and muscle tissue samples from the same aquaculture pond of *Procambarus clarkii* were randomly collected and flash-frozen in liquid nitrogen for genomic DNA extraction. Real-time fluorescence qPCR was used to detect the white spot syndrome virus copy number in the gill tissue of each shrimp using primers WDP07: 5'-ACAACTAGCAAGCACTGGTAT-3' and WDP06: 5'-TCAGCTTCAAGAATCCCAATAG-3'. The results are as follows: Figure 1 As shown in Table 1. Then, 15 individuals with extremely low viral copy numbers and 15 individuals with extremely high viral copy numbers were selected to form two gene pools. The viral copy numbers of these two pools are shown in Table 1. Table 1 shows the sequencing population of the red swamp crayfish in the mixed pools. Individuals 1-15 belong to the group with extremely low viral copy numbers, and individuals 16-30 belong to the group with extremely high viral copy numbers.

[0030] Table 1. Virus carriage status in the mixed-pool sequencing population.

[0031]

[0032]

[0033] 2. BSA resequencing analysis

[0034] DNA was extracted from muscle tissue from the two populations mentioned above, and high and low viral copy number pools were constructed by mixing equal amounts of DNA. After the DNA samples passed the tests, the genomic DNA was fragmented using ultrasound to construct a small DNA fragment library. High-throughput sequencing of the high and low viral copy number pools was performed using an Illumina Hiseq 2500 sequencer at a sequencing depth of 30X. The mutation analysis software GATK was used to detect population SNPs, and further filtering was performed based on factors such as quality value, depth, and reproducibility to finally obtain high-confidence variant sites. The detected candidate SNPs were annotated using ANNOVAR software and the published genome annotation file (GCF_040958095.1). By screening SNP-index differential regions, candidate regions of the research target trait were obtained, completing the initial localization of the target SNPs.

[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. Comparison revealed that two segments of the TRIM gene may contain SNP sites related to WSSV tolerance.

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

[0037] Based on the full-length cDNA sequence information of the TRIM gene of Procambarus clarkii (XP_045581296) reported in Genbank, a Blast search was performed on its amino acid coding sequence. By comparing the Blast results, the distribution of introns and exons and the 5'-flanking regulatory region of the TRIM gene of Procambarus clarkii were predicted.

[0038] The full-length TRIM gene of the red swamp crayfish is 12691 bp, including 4 exons and 3 introns; extron1 is 212 bp, extron2 is 218 bp, extron3 is 100 bp, and extron4 is 7077 bp; introns intron1 is 1989 bp, intron2 is 2239 bp, intron3 is 2856 bp, and there is a 1000 bp 5'-flanking regulatory region. Sequence structure information can be found in [link to generic information]. Figure 2 .

[0039] Example 2: Screening of TRIM gene SNPs in Procambarus clarkii and its correlation with WSSV tolerance trait.

[0040] 1. Test Methods

[0041] 1.1 Separation of sensitive and tolerant groups

[0042] The *Procambarus clarkii* samples used in this experiment were obtained from the *Procambarus clarkii* Breeding and Selection Base of Huazhong Agricultural University (Caidian). Before the experiment, the shrimp were temporarily housed in polyethylene plastic culture tanks for 3 days, and 300 healthy and vigorous shrimp were selected for the experiment. The indoor culture water temperature was 24-25℃. During the experiment, the shrimp were fed commercial feed once a day, and the water was changed every two days. 0.1 mL of WSSV (10...) was injected into each of the 300 healthy *Procambarus clarkii* shrimp. 5 (Copy), the injection site is the second abdominal segment. As the virus replicates and spreads within the shrimp, the first 50 swamp crayfish that become ill and die are considered a susceptible population. After 15 days, 50 surviving swamp crayfish are considered a tolerant population.

[0043] 1.2 Extraction of Genomic DNA

[0044] Tail muscle samples from two populations of Procambarus clarkii were collected and stored in 2 mL centrifuge tubes containing anhydrous ethanol. DNA was extracted from the samples according to the kit instructions, and the resulting DNA samples were stored at -20°C.

[0045] 1.3 Primer Design

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

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

[0048]

[0049] 1.4 PCR amplification

[0050] Using DNA from the tolerant and susceptible groups of *Procambarus clarkii* as templates, PCR amplification was performed using the primers shown in Table 2. The amplification system (Kangwei Century) was: 25 μL of 2×Es Taq MasterMix (Dye), 2 μL each of upstream and downstream primers (10 nM), 1 μL of DNA template (100 ng / L), and ddH2O to bring the volume to 50 μL. The PCR reaction consisted of 35 cycles, with pre-denaturation at 94℃ for 2 min. Each cycle included denaturation at 94℃ for 30 s, annealing at 56℃ for 30 s, and extension at 72℃ for 30 s. After each cycle, a final extension at 72℃ for 2 min was performed.

[0051] 1.5 Small-scale SNP screening and large-scale SNP validation

[0052] Twenty individuals were randomly selected from both the tolerant and susceptible groups. PCR amplification of DNA from both groups was performed using designed primers. Two μL of the amplification product was analyzed by 1.5% agarose gel electrophoresis. PCR products with clear and bright target bands were sent to Wuhan HeCe Gene Technology Co., Ltd. for sequencing. The sequencing results were analyzed using SnapGene software to examine the nucleotide sequence and compare sequencing peaks. Sites showing significant differences between the tolerant and susceptible groups in a small population were validated in individual samples from the remaining 60 individuals.

[0053] 1.6 Data Analysis

[0054] The frequencies of each SNP genotype on TRIM gene fragment 1 and fragment 2 were calculated separately, and the chi-square test was performed on each SNP genotype and tolerance trait using SPSS 22.0.

[0055] 2. Results Analysis

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

[0057] Amplification and sequencing of TRIM gene fragments 1 and 2 of Procambarus clarkii were performed. After comparison, a total of 9 SNP sites were found in fragments 1 and 2. One SNP site with significant differences between the tolerant and sensitive groups was found in fragment 2. 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 of 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 of TRIM gene SNP sites and WSSV tolerance traits in Procambarus clarkii

[0064] The genotype frequencies of the five SNP loci in fragment 2 were statistically analyzed in the tolerant and sensitive groups. The chi-square test in SPSS 22.0 software was used to test the significance of the differences in the genotype frequencies of these five SNP loci between the tolerant and sensitive groups. P < 0.05 was considered to be significant, and otherwise no significant difference was found.

[0065] Table 4. Association analysis of 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 the g.3304T>C group, the proportion of TC genotype Procambarus clarkii in the tolerant group was higher than that in the sensitive group. Chi-square test showed a significant difference in the frequency of the g.3304T>C genotype between the tolerant and sensitive groups (P<0.05). These results indicate a significant correlation between g.3304T>C and WSSV tolerance, and that g.3304T>C is the dominant genotype in the WSSV tolerant group. Therefore, WSSV tolerance breeding can be carried out by selecting Procambarus clarkii with the TC genotype at the g.3304T>C site of the TRIM gene.

[0070] References

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[0073] 3. Luo Xiaohong. Seasonal variation dynamics of white spot syndrome virus (WSSV) and internal microbial community in red swamp crayfish under different farming models [D]. Hubei: Huazhong Agricultural University, 2023.

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

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Claims

1. A method for breeding varieties of red swamp crayfish resistant to white spot syndrome virus for non-disease diagnostic purposes, characterized in that, Includes the following steps: 1) Extract genomic DNA from the red swamp crayfish; 2) Using the extracted genomic DNA as a template, design PCR primers to amplify the DNA fragment with the nucleotide sequence shown in SEQ ID NO.1; 3) Sequencing the PCR amplification products to detect the genotype of the SNP molecular marker, which is located at the 585th bp of the DNA sequence shown in SEQ ID NO.1, with the bases being T or C. The T / C heterozygous genotype is a favorable marker for resistance to white spot syndrome virus in Procambarus clarkii.

2. The breeding method for red swamp crayfish resistant to white spot syndrome virus as described in claim 1, characterized in that: The sequences of the PCR primers are shown in SEQ ID NO.2 and SEQ ID NO.3, which are the upstream and downstream primers for amplifying the DNA fragment shown in SEQ ID NO.1, respectively.

3. The method for breeding varieties of red swamp crayfish resistant to white spot syndrome virus as described in claim 1, characterized in that, The PCR amplification system was as follows: 25 μL of 2×Es Taq MasterMix (Dye), 2 μL each of 10 nM upstream and downstream primers, 1 μL of 100 ng / L DNA template, and ddH2O to bring the volume to 50 μL.

4. The method for breeding varieties of red swamp crayfish resistant to white spot syndrome virus as described in claim 1, characterized in that, The PCR amplification program is as follows: pre-denaturation at 94℃ for 2 min, each cycle includes denaturation at 94℃ for 30 s, annealing at 56℃ for 30 s, extension at 72℃ for 30 s, for a total of 35 cycles; after the cycle, extension at 72℃ for 2 min.

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