SNP loci, locus combinations and their applications related to resistance to iridovirus disease in Macrobrachium rosenbergii

Abstract

The present invention discloses SNP sites, site combinations and applications related to the resistance to iridovirus disease of Macrobrachium rosenbergii, and belongs to the field of aquatic biological breeding. The present invention successfully screened out SNP sites related to the resistance to iridovirus disease of Macrobrachium rosenbergii through whole genome association analysis, which were located at bases 9574545 of chromosome 10 of Macrobrachium rosenbergii and bases 26491028 of chromosome 14 of Macrobrachium rosenbergii, respectively. For the two SNP sites, a combination of SNP sites related to the resistance to iridovirus disease of Macrobrachium rosenbergii was further developed, and by detecting the combination of SNP sites, individuals with resistance to iridovirus disease of Macrobrachium rosenbergii can be screened. Therefore, the SNP molecular markers provided by the present invention can be used for molecular marker-assisted breeding of Macrobrachium rosenbergii, accelerate the breeding of disease-resistant Macrobrachium rosenbergii varieties, and provide a reference basis for the disease-resistant breeding of Macrobrachium rosenbergii.

Description

Technical Field

[0001] The present invention relates to the field of aquatic biological breeding, and in particular to SNP sites and site combinations and applications thereof associated with Macrobrachium rosenbergii iridovirus disease resistance. Background Art

[0002] Macrobrachium rosenbergii Macrobrachium rosenbergii ) belongs to the order Decapoda, family Palaemonidae, genus Macrobrachium Macrobrachium ), is one of the important freshwater aquaculture species in my country. It is widely popular in the aquatic consumer market due to its excellent meat quality and high economic value. However, in recent years, due to the high-density and intensive farming model, the outbreak of iridovirus disease in the farming process of Macrobrachium rosenbergii has posed a serious threat to the industry.

[0003] Iridovirus disease, caused by Decapoda Iridovirus 1 (DIV1), is a highly contagious disease. Sick Macrobrachium rosenbergii exhibits a variety of clinical symptoms, including loss of appetite, slow movement, weak swimming, slow swimming, lying on the side, and the most typical "white head" phenomenon - obvious white spots appear inside the carapace at the base of the frontal sword. As the disease worsens, the body color of the diseased shrimp gradually turns reddish, the gills become congested and red, the hepatopancreas atrophies, the jejunum becomes empty, and eventually leads to large-scale deaths. These symptoms not only seriously affect the growth rate and health of the shrimp, but also lead to large-scale deaths, causing huge economic losses to farmers.

[0004] SNP (single nucleotide polymorphism) is one of the most common forms of genetic variation, which can reveal genetic differences between different individuals and provide an important basis for the study of genetic traits. As an advanced genetic tool, genome-wide association analysis (GWAS) can identify the complex relationship between genes and specific phenotypes (such as disease resistance) by utilizing the linkage disequilibrium phenomenon between SNPs.

[0005] In the field of genetic breeding for disease resistance in plants and livestock, GWAS has achieved remarkable results. By mining molecular markers related to major selected traits (such as disease resistance) and applying them to marker-assisted selection (MAS) and whole genome selection (GS), scientists have successfully bred varieties with multiple excellent traits. Compared with traditional breeding methods, these technologies not only accelerate the process of pure and homozygous alleles related to traits, but also greatly improve breeding efficiency and genetic selection progress, providing strong support for the sustainable development of agricultural production.

[0006] In the field of genetic breeding for disease resistance in plants and livestock, the use of molecular information to assist breeding has been widely used. However, in the field of disease resistance breeding in aquatic animals, especially in the breeding of iridovirus disease resistance in Macrobrachium rosenbergii, the application of GWAS is still in its infancy. Although there have been many reports on genetic variation and disease resistance in Macrobrachium rosenbergii in recent years, there has been no detailed study on SNP loci associated with iridovirus disease resistance.

[0007] Therefore, identifying and verifying these key SNP sites will provide new possibilities and directions for disease-resistant breeding of Macrobrachium rosenbergii and sustainable development of the industry. Summary of the invention

[0008] The present invention aims to provide a reagent for detecting the 9574545th base of chromosome 10 of Macrobrachium rosenbergii and its application in breeding of Macrobrachium rosenbergii resistant to iris virus disease.

[0009] Another object of the present invention is to provide a reagent for detecting the 9574545th base of chromosome 10 of Macrobrachium rosenbergii and a reagent for detecting the 26491028th base of chromosome 14 of Macrobrachium rosenbergii for use in breeding of Macrobrachium rosenbergii resistant to iris virus disease.

[0010] In order to achieve the above object, the present invention adopts the following technical measures:

[0011] A large-scale DIV1 challenge experiment with Macrobrachium rosenbergii was conducted. The dead and surviving samples of the challenge experiment were selected. The magnetic bead method was used to extract the sample DNA, and the quality of the extracted DNA was detected by agarose gel electrophoresis. The samples that passed the quality control were sent to Huazhi Biotechnology Co., Ltd. for sequencing using a liquid phase chip of Macrobrachium rosenbergii. The software GATK was used for genotyping, and beagle was used to fill in the missing genotypes. Plink was used to perform quality control filtering on the typing file to obtain the final typing results. After quality control of the sequencing data and alignment to the reference genome, GATK was used for variation detection. VCFtools was used to filter the SNP data for site deletion rate and minimum allele frequency to obtain the final typing results. The disease resistance phenotype was in the survival-death state case-control format. The phenotype and genotype were combined using the GEMMA software for genome-wide association analysis (GWAS), and the threshold was set to -log 10 (1e-05)=5, where 1e-05 means 1×10 -5, the sites with p-value reaching the threshold have significant correlation. Finally, two SNPs related to iridovirus resistance were screened out. The SNP site Chr10-9574545 is located at the 300th position in SEQ ID No.1, where the base Y at the 300th position is A (adenine) or G (guanine); the SNP site Chr14-26491028 is located at the 300th position of the sequence displayed in SEQ ID No.2, where the base W at the 300th position is A (adenine) or T (thymine).

[0012] The sequence shown in SEQ ID No.1 is:

[0013] , where Y is A or G.

[0014] The sequence shown in SEQ ID No.2 is:

[0015] , where W is A or T.

[0016] The invention provides application of a reagent for detecting the 9574545th base of chromosome 10 of Macrobrachium rosenbergii in breeding of Macrobrachium rosenbergii resistant to iris virus disease.

[0017] The present invention also provides the use of a reagent for detecting the 9574545th base of chromosome 10 of Macrobrachium rosenbergii and a reagent for detecting the 26491028th base of chromosome 14 of Macrobrachium rosenbergii in breeding of Macrobrachium rosenbergii resistant to iris virus disease.

[0018] In the above-mentioned application, preferably, the reagent is a primer. In the present application, the SNP can be detected by conventional PCR primers, so those skilled in the art can detect the SNP site by conventional primer design.

[0019] Preferably, the reagent is a primer, which is: Chr10-9574545: upstream primer F: 5'-AATCTTTATGGAAACTTGGAGTG-3', downstream primer R: 5'-CAAGTAGCAGTATCAGCAGTAGTG-3'.

[0020] Preferably, the reagents are primers, namely: Chr10-9574545: upstream primer F: 5'-AATCTTTATGGAAACTTGGAGTG-3', downstream primer R: 5'-CAAGTAGCAGTATCAGCAGTAGTG-3' and Chr14-26491028: upstream primer F: 5'-GACAAAATCATTTGAATATCTCTGT-3', downstream primer R: 5'-TCTCTAATGGAGCTCATGCTGT-3'.

[0021] The present invention also provides a detection primer for SNP marker of iridovirus disease resistance-associated gene of Macrobrachium rosenbergii, comprising the following primers:

[0022] Chr10-9574545: upstream primer F: 5′-AATCTTTATGGAAACTTGGAGTG-3′, downstream primer R: 5′-CAAGTAGCAGTATCAGCAGTAGTG-3′;

[0023] The SNP marker is located at the 300th base from the 5' end of the sequence shown in SEQ ID NO.1, and the base is A or G.

[0024] The present invention also provides a detection primer for SNP marker of iridovirus disease resistance-associated gene of Macrobrachium rosenbergii, comprising the following primers:

[0025] Chr10-9574545: upstream primer F: 5′-AATCTTTATGGAAACTTGGAGTG-3′, downstream primer R: 5′-CAAGTAGCAGTATCAGCAGTAGTG-3′ and Chr14-26491028: upstream primer F: 5′-GACAAAATCATTTGAATATCTCTGT-3′, downstream primer R: 5′-TCTCTAATGGAGCTCATGCTGT-3′;

[0026] The SNP marker is located at the 300th base from the 5' end of the sequence shown in SEQ ID NO.1, and the base is A or G;

[0027] The SNP marker is located at the 300th base from the 5' end of the sequence shown in SEQ ID NO.2, and the base is A or T.

[0028] The present invention also provides a method for breeding a variety of Macrobrachium rosenbergii resistant to decapod iridovirus disease, comprising the following steps:

[0029] a. Extracting the genomic DNA of Macrobrachium rosenbergii to be tested;

[0030] b. performing PCR amplification on the genomic DNA of the Macrobrachium rosenbergii to be tested using the above-mentioned detection primer or detection primer combination;

[0031] c. Sequence the amplified product to determine the genotype of the above-mentioned SNP marker and select individuals with AG genotype, or determine the genotype of the above-mentioned SNP marker and select individuals with AG genotype and / or TT genotype as reserve parents for breeding of Macrobrachium rosenbergii resistant to iris virus disease.

[0032] Beneficial effects of the present invention:

[0033] Compared with the prior art, the beneficial results of the present invention are:

[0034] The present invention screened out two SNP molecular markers that are significantly associated with the resistance to iridovirus disease of Macrobrachium rosenbergii. The relevant SNP molecular markers can be used for molecular marker-assisted breeding of Macrobrachium rosenbergii, accelerate the selection of disease-resistant Macrobrachium rosenbergii varieties, and provide a reference basis for the disease-resistant breeding of Macrobrachium rosenbergii or other cultured shrimps. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Manhattan plot of genome-wide association analysis between disease-resistant and susceptible groups. DETAILED DESCRIPTION

[0036] In order to enable those skilled in the art to better understand the technical content of the present invention, the present invention is further described below in conjunction with specific embodiments and drawings. The technical solutions described in the present invention, unless otherwise specified, are conventional solutions in the art; the reagents or materials, unless otherwise specified, are all from commercial channels.

[0037] Example 1

[0038] Mining of SNPs associated with resistance to iridovirus disease in Macrobrachium rosenbergii.

[0039] A large-scale Macrobrachium rosenbergii decapod iridovirus 1 challenge experiment was conducted. The experimental shrimp weighed 1.5±0.2 g. 500 dead samples from the challenge experiment were selected as the susceptible group, and 498 surviving samples were selected as the disease-resistant group. The sample DNA was extracted by magnetic beads, and the concentration of the DNA sample was detected by Qubit fluorescence quantification instrument. The integrity of the DNA was detected by 1% agarose gel electrophoresis. The samples that passed the quality control were used for library construction and quality control. The samples that passed the quality control were sent to Huazhi Biotechnology Co., Ltd. for sequencing using liquid phase chip of Macrobrachium rosenbergii. The software GATK was used for genotyping, and beagle was used to fill in the missing genotypes. Plink was used to filter the typing files for quality control to obtain the final typing results.

[0040] After quality control of the sequencing data and alignment to the reference genome, GATK was used for variant detection. VCFtools was used to filter the SNP data for site deletion rate and minimum allele frequency to obtain the final typing results. The disease resistance phenotype was in the survival-death state case-control format. GEMMA software was used to combine phenotypes and genotypes for genome-wide association analysis (GWAS), with the threshold set to -log 10 (1e-05)=5, the sites with p-value reaching the threshold have significant correlation. Use ggplot2 package to create Manhattan plot as shown below Figure 1 shown.

[0041] The results showed that there were 10 SNP loci among all SNPs that met the requirements. Through Blast analysis and comparison, two SNPs that may be related to the resistance of Macrobrachium rosenbergii to iridovirus disease were preliminarily identified based on physical information and gene annotations in the linkage region.

[0042] The SNP site Chr10-9574545 is located at the 300th position in SEQ ID No.1, wherein the base Y at the 300th position is A (adenine) or G (guanine); the SNP site Chr14-26491028 is located at the 300th position of the sequence displayed in SEQ ID No.2, wherein the base W at the 300th position is A (adenine) or T (thymine).

[0043] Example 2

[0044] Application of SNP loci associated with resistance to iridovirus disease in Macrobrachium rosenbergii breeding.

[0045] Based on the two SNP sites located in Example 1 of the present invention, corresponding primers were developed for molecular marker-assisted selection breeding of disease-resistant Macrobrachium rosenbergii to obtain Macrobrachium rosenbergii varieties resistant to iris virus disease. The primer information is shown in Table 1.

[0046] Table 1 Nucleotide sequences of primers for amplifying SNPs associated with iridovirus resistance in Macrobrachium rosenbergii

[0047]

[0048] About 200 healthy Macrobrachium rosenbergii were selected from the breeding pond of Zhejiang Lanke Seed Technology Co., Ltd. for the decapod iridovirus 1 challenge experiment, 75 diseased and dead individuals and 75 disease-resistant surviving individuals were collected, and the genotype of the two SNP sites located in Example 1 was detected to verify the correlation between the genotype and the resistance of Macrobrachium rosenbergii to iridovirus disease. Specifically, the following steps were included:

[0049] The muscle DNA of 150 tails of the validation group was extracted by magnetic bead method and diluted to 100 ng / μL for later use. The genomic DNA of Macrobrachium rosenbergii was used as template for PCR amplification.

[0050] The PCR amplification program for detecting the Chr10-9574545 and Chr14-26491028 sites of Macrobrachium rosenbergii was as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing for 30 s, extension at 72°C for 40 s, 34 cycles; extension at 72°C for 10 min, and storage at 4°C.

[0051] The PCR amplification system for detecting the Chr10-9574545 and Chr14-26491028 loci of Macrobrachium rosenbergii was as follows: 1.0 μL of upstream and downstream primers, 1.0 μL of DNA template, 12.5 μL of PCR Master Mix (Sangon, Shanghai, China), and ddH2O to 25 μL.

[0052] 2) Genotype statistics

[0053] After PCR was completed, Sanger sequencing was used to obtain the sequence of the corresponding samples, and the genotypes of the Chr10-9574545 and Chr14-26491028 loci of Macrobrachium rosenbergii were read according to the sequencing peak graph of each sample, and the survival rate and mortality rate were calculated. The statistical results are shown in Table 2.

[0054] Table 2 Statistical analysis of disease resistance SNP genotypes in a validation population of 400 Macrobrachium rosenbergii

[0055]

[0056] From Table 2, we can see that among the 140 diseased Macrobrachium rosenbergii, there are 83 individuals with the genotype of Chr10-9574545 AG, of which 49 survived and 34 died; there are 62 individuals with the genotype of Chr14-26491028 TT, of which 40 survived and 22 died. Therefore, in Macrobrachium rosenbergii, if the genotype of the individual at the genomic SNP site Chr10-9574545 is AG, or the genotype of the individual at the SNP site Chr14-26491028 is TT, the probability of survival of the individual suffering from iridovirus disease is higher than the probability of death. The two can also be used at the same time, that is, when the genotype of the individual's SNP site Chr10-9574545 is AG and the genotype of the Chr14-26491028 site is TT, the survival rate is significantly higher than the mortality rate, that is, the individual is iridovirus disease-resistant Macrobrachium rosenbergii.

[0057] The above embodiments are only used to illustrate the present invention, and the protection scope of the present invention is not limited to the above embodiments. A person skilled in the art can achieve the purpose of the present invention based on the above disclosure of the present invention, and any improvement and deformation based on the concept of the present invention shall fall within the protection scope of the present invention, and the specific protection scope shall be subject to the claims.

Claims

1. Application of a reagent for detecting SNP molecular markers on chromosome 10 of Macrobrachium rosenbergii in breeding of Macrobrachium rosenbergii resistance to iris virus disease, wherein the sequence of the SNP molecular marker on chromosome 10 of Macrobrachium rosenbergii is shown in SEQ ID NO.1, and the SNP molecular marker has an A / G mutation at position 300, and the application is to select individuals with the SNP molecular marker AG genotype for breeding of Macrobrachium rosenbergii resistance to iris virus disease.

2. Use of a reagent for detecting SNP molecular markers on chromosome 10 of Macrobrachium rosenbergii and a reagent for detecting SNP molecular markers on chromosome 14 of Macrobrachium rosenbergii in breeding of Macrobrachium rosenbergii for resistance to iris virus disease, wherein the sequence of the SNP molecular marker on chromosome 10 of Macrobrachium rosenbergii is shown in SEQ ID NO.1, and the SNP molecular marker on chromosome 10 of Macrobrachium rosenbergii has an A / G mutation at position 300, and the sequence of the SNP molecular marker on chromosome 14 of Macrobrachium rosenbergii is shown in SEQ ID NO.2, and the SNP molecular marker on chromosome 14 of Macrobrachium rosenbergii has an A / T mutation at position 300, The application is to select individuals whose SNP molecular marker of chromosome 10 is AG genotype and whose SNP molecular marker of chromosome 14 is TT genotype for breeding of Macrobrachium rosenbergii resistant to iris virus disease.

3. According to the use according to claim 1 or 2, the reagent is a primer.

4. The use according to claim 1, wherein the reagent is a primer, and the primer is a detection primer Chr10-9574545 for detecting the SNP molecular marker of chromosome 10 of Macrobrachium rosenbergii: upstream primer F: 5'-AATCTTTATGGAAACTTGGAGTG-3', downstream primer R: 5'-CAAGTAGCAGTATCAGCAGTAGTG-3'.

5. The use according to claim 2, wherein the reagent is a primer, and the primer is a detection primer Chr10-9574545 for detecting the SNP molecular marker on chromosome 10 of Macrobrachium rosenbergii: upstream primer F: 5'-AATCTTTATGGAAACTTGGAGTG-3', downstream primer R: 5'-CAAGTAGCAGTATCAGCAGTAGTG-3' and a detection primer Chr14-26491028 for detecting the SNP molecular marker on chromosome 14 of Macrobrachium rosenbergii: upstream primer F: 5'-GACAAAATCATTTGAATATCTCTGT-3', downstream primer R: 5'-TCTCTAATGGAGCTCATGCTGT-3'.

6. A method for breeding Macrobrachium rosenbergii varieties resistant to iris virus disease, characterized in that: The following steps are involved: a. Extracting the genomic DNA of Macrobrachium rosenbergii to be tested; b. PCR amplification of the genomic DNA of Macrobrachium rosenbergii to be tested is performed using the detection primers of the SNP marker of the gene associated with the iridovirus disease resistance trait of Macrobrachium rosenbergii; The detection primer is any one of the following: (1) Detection primers for detecting the SNP molecular marker on chromosome 10 of Macrobrachium rosenbergii Chr10-9574545: upstream primer F: 5'-AATCTTTATGGAAACTTGGAGTG-3', downstream primer R: 5'-CAAGTAGCAGTATCAGCAGTAGTG-3'; (2) The detection primers Chr10-9574545 for detecting the SNP molecular marker on chromosome 10 of Macrobrachium rosenbergii: upstream primer F: 5'-AATCTTTATGGAAACTTGGAGTG-3', downstream primer R: 5'-CAAGTAGCAGTATCAGCAGTAGTG-3' and the detection primers Chr14-26491028 for detecting the SNP molecular marker on chromosome 14 of Macrobrachium rosenbergii: upstream primer F: 5'-GACAAAATCATTTGAATATCTCTGT-3', downstream primer R: 5'-TCTCTAATGGAGCTCATGCTGT-3'; The sequence of the SNP molecular marker of chromosome 10 of Macrobrachium rosenbergii is shown as SEQ ID NO.1, and the SNP molecular marker of chromosome 10 of Macrobrachium rosenbergii has an A / G mutation at position 300; the sequence of the SNP molecular marker of chromosome 14 of Macrobrachium rosenbergii is shown as SEQ ID NO.2, and the SNP molecular marker of chromosome 14 of Macrobrachium rosenbergii has an A / T mutation at position 300; c. Sequence the amplified products to determine the genotype of the SNP marker of chromosome 10, and select individuals with the AG genotype, or determine the genotypes of the SNP markers of chromosomes 10 and 14, and select individuals with the SNP molecular marker of chromosome 10 as the AG genotype and the SNP molecular marker of chromosome 14 as the TT genotype as reserve parents for breeding varieties of Macrobrachium rosenbergii resistant to iris virus disease.

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