Probe for detecting SNP molecular marker combination of Japanese prawn and 20K liquid chip and application thereof

Abstract

This invention discloses a probe for detecting SNP molecular marker combinations in Litopenaeus vannamei and a 20K liquid phase chip, as well as their applications, belonging to the field of molecular breeding technology. This invention screened and obtained 20,224 high-quality, highly polymorphic, and chromosome-uniformly distributed SNP loci. Probes were designed targeting these 20,224 SNP markers, thereby obtaining the Litopenaeus vannamei 20K liquid phase chip. This invention also discloses the application of the Litopenaeus vannamei 20K liquid phase chip in Litopenaeus vannamei breeding, sample detection, genome-wide association analysis, genome selection, and disease resistance in Litopenaeus vannamei. The Litopenaeus vannamei 20K liquid phase breeding chip provided by this invention is a precise and efficient molecular breeding chip with advantages such as high specificity, high marker density, strong automation, and high detection throughput. It can be fully used for SNP genotyping detection of different Litopenaeus vannamei samples, can establish a modern breeding system, promote the development of the Litopenaeus vannamei breeding industry, and has significant promotional value.

Description

Technical Field

[0001] This invention belongs to the field of molecular breeding technology, specifically relating to a probe for detecting SNP molecular marker combinations in Japanese shrimp and a 20K liquid phase chip and their applications. Background Technology

[0002] Japanese shrimp ( Marsupenaeus japonicus Also known as the Japanese prawn, it belongs to the order Decapoda, family Penaeidae, and genus Penaeus. It has a very wide distribution, and breeding superior varieties is of great significance to the Japanese prawn aquaculture industry. On the one hand, since Japanese prawns are mainly farmed in the coastal waters of my country, the water used for farming is almost entirely drawn from natural seawater. Pathogens such as Vibrio are widely present in the seawater of estuaries, bays, and nearshore waters, and can easily come into contact with farmed Japanese prawns through water bodies, sediments, or plankton. Therefore, disease is an unavoidable problem in the Japanese prawn aquaculture industry, and disease resistance has become an important stress-resistance trait pursued by the Chinese Japanese prawn pond aquaculture industry. At the same time, in the Japanese prawn aquaculture industry, the time from onset to death is generally only a dozen hours, and in severe cases, the cumulative mortality rate of Japanese prawns in affected ponds can reach over 90% within 1-2 days. Currently used drugs and antibiotics are not ideal for treating Japanese prawn diseases. In actual farming, once a disease occurs, farmed Japanese prawns will die in large numbers, causing huge economic losses to farmers. Therefore, the breeding of disease-resistant varieties is also an urgent need for the Japanese shrimp farming industry.

[0003] Traditional breeding methods rely on phenotypic selection, which has disadvantages such as long cycles and instability. Molecular breeding refers to the technique of selecting breeding materials using DNA molecular markers. Molecular breeding methods select reserve parents based on effective molecular markers, which can more quickly improve the economic traits of offspring. Currently, commonly used molecular marker identification methods include RFLP, RAPD, and SSR technologies, but these methods all have certain drawbacks. For example, RFLP experiments are cumbersome, have long detection cycles, and are expensive, making them unsuitable for large-scale molecular breeding; RAPD technology is easily affected by external factors, resulting in unstable results and poor reproducibility. Therefore, developing molecular markers related to disease resistance traits is of great significance for the healthy aquaculture of Japanese shrimp and accelerating the breeding process. Summary of the Invention

[0004] Based on the above requirements, the purpose of this invention is to provide a probe and a 20K liquid phase chip for detecting SNP molecular marker combinations in Japanese shrimp, and their applications. This invention provides a liquid phase chip developed and prepared from 20,224 SNP molecular markers. This 20K liquid phase chip is a precise and efficient molecular breeding chip, possessing characteristics such as high specificity, high marker density, strong automation, and high detection throughput, and can be widely applied to the detection of different varieties of Japanese shrimp.

[0005] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a probe for detecting SNP molecular marker combinations in Japanese shrimp, wherein the Japanese shrimp SNP molecular marker combination consists of 20,224 SNP molecular markers; the 20,224 SNP molecular markers are shown in Table 1, and the location and variation information of the SNP sites are represented in the form of chromosome: physical location: reference genotype / variant allele.

[0006] Furthermore, the site information of the 20,224 SNP molecular markers is based on the NCBI Japanese shrimp reference genome (https: / / www.ncbi.nlm.nih.gov / Traces / wgs / JBUDMT01?display=contigs).

[0007] This invention also provides a 20K liquid phase chip for Japanese shrimp, which includes probes or primers for detecting SNP molecular marker combinations in Japanese shrimp. The SNP molecular marker combinations in Japanese shrimp consist of 20,224 SNP molecular markers. The 20,224 SNP molecular markers are shown in Table 1. The location and variation information of the SNP sites are represented in the form of chromosome: physical location: reference genotype / variant allele.

[0008] Furthermore, the 20,224 SNP molecular markers were developed into a liquid phase chip for Japanese shrimp using targeted sequencing genotyping technology.

[0009] The present invention also provides the application of the probe or the 20K liquid phase chip for Japanese shrimp in molecular marker-assisted breeding of Japanese shrimp.

[0010] The present invention also provides the application of the probe or the 20K liquid phase chip for Japanese shrimp in the genotyping detection of Japanese shrimp.

[0011] Furthermore, the genotyping detection steps include: (1) Extract genomic DNA from the Japanese shrimp samples to be tested; (2) The genomic DNA was digested with a fragmentation enzyme and an A base was added to the 3' end. The sequencing adapter and the DNA fragment were ligated using T4 ligase. The ligation product was purified. The purified ligation product was amplified by PCR. The amplified product was then screened for fragments to construct a library. (3) After adding Japanese shrimp 20K liquid phase chip and hybridization reagent to the library, target segment capture is performed, followed by a round of PCR amplification to construct cGPS sequencing library; (4) Perform data analysis on the cGPS sequencing library and perform mutation site analysis to obtain the original vcf mutation result file; when the proportion of mutation reads supporting the mutation is ≥0.8 or ≤0.2, the site is judged as homozygous genotype; when the proportion of mutation reads supporting the mutation is between 0.2 and 0.8, the site is judged as heterozygous genotype.

[0012] The present invention also provides the application of the probe or the Japanese shrimp 20K liquid phase chip in QTL localization analysis of target traits in Japanese shrimp.

[0013] The present invention also provides the application of the probe or the Japanese shrimp 20K liquid phase chip in genome-wide association analysis of Japanese shrimp.

[0014] The present invention also provides the application of the probe or the Japanese shrimp 20K liquid phase chip in the discovery or identification of disease resistance genes in Japanese shrimp.

[0015] The present invention also provides the application of the probe or the Japanese shrimp 20K liquid phase chip in the identification of kinship in Japanese shrimp.

[0016] This invention also provides the application of the aforementioned liquid phase chip for Japanese shrimp in the identification, improvement, and genetic diversity analysis of Japanese shrimp germplasm resources.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The Japanese shrimp 20K liquid phase breeding chip provided by this invention has the advantages of good site representativeness, strong specificity and high polymorphism. It is a precise and efficient molecular breeding chip that can be widely used for the detection of different varieties of Japanese shrimp materials.

[0018] 2. The Japanese shrimp 20K liquid phase breeding chip of the present invention is based on high-throughput sequencing technology. This detection method has high detection throughput and produces a large amount of data at one time, which can simultaneously cover the detection of nearly a thousand materials. It is also applicable to mainstream second-generation sequencing platforms such as Illumina and MGI, and has broad platform adaptability.

[0019] 3. The 20K liquid phase breeding chip for Japanese shrimp of the present invention can be used for molecular marker analysis of Japanese shrimp varietal resources, genotyping of offspring of Japanese shrimp hybrid populations, identification of the authenticity of Japanese shrimp varietals, genetic background analysis and screening of Japanese shrimp breeding materials, genome-wide association analysis of Japanese shrimp, genetic diversity analysis of germplasm resources, and identification of kinship.

[0020] 4. The Japanese shrimp 20K SNP chip provided by this invention has a high target site detection rate, good stability, and accurate and reliable typing results when performing genotyping on the material to be tested. It can be fully used for SNP typing detection of different Japanese shrimp samples. Attached Figure Description

[0021] Figure 1 This is a chromosome distribution map of the 20K locus in Japanese shrimp.

[0022] Figure 2 The detection rate of loci in 20K liquid phase chip samples of Japanese shrimp.

[0023] Figure 3 The heterozygosity of loci in 20K liquid phase chip samples of Japanese shrimp.

[0024] Figure 4 Manhattan plot of the location. Detailed Implementation

[0025] The technical solution of the present invention will be further described in detail with reference to the following specific examples, but the scope of protection claimed by the present invention is not limited to the scope described in the examples.

[0026] The present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0027] Example 1: Development of a 20K liquid phase chip for Japanese shrimp This embodiment provides a 20K SNP liquid phase chip for Japanese shrimp, which includes 20,224 molecular markers. The site information of the 20,224 SNP molecular markers is shown in Table 1.

[0028] The aforementioned 20K liquid phase chip for Japanese shrimp is obtained through the following method: Based on whole-genome data from 96 Japanese shrimp samples, quality indicators for resequencing sites were calculated. Polymorphic SNP sites located in gene regions and meeting the following quality indicators for each population—minimum allele frequency (MAF) ≥ 0.1, SNP detection rate ≥ 0.9, deletion rate ≤ 0.1, heterozygosity rate ≤ 0.4, and sequencing depth ≥ 10X—were selected as initial candidate sites. The location of the SNPs on the gene was also considered, and the retention order was as follows: exonic > splicing > UTR > intronic > upstream > downstream > intergenic—to ensure that the microarray sites could cover all genes on the reference genome as much as possible.

[0029] All candidate and functional sites were used to design probes. The probe design method was as follows: for each site, probes were designed within a 100 bp range upstream and downstream, with a probe length of approximately 100 bp and a GC content between 20% and 80%. Probes that could not be uniquely aligned to the genome or contained repetitive sequences in their flanking sequences were removed. Based on the principle of uniform distribution of physical locations, SNP sites uniformly distributed on the chromosomes of Japanese shrimp were screened.

[0030] Using the above methods, 20,224 highly polymorphic and high-quality SNP loci were finally obtained through screening. These loci represent abundant gene regions with a gene coverage rate as high as 95%. The loci are evenly distributed on the chromosome, with an average coverage rate of 99.61% and an average distance between loci of 75,419 bp. The distribution of the loci on the chromosome is shown in the figure below. Figure 1 The final 20,224 SNP sites were selected, and the site information of the 20,224 SNP molecular markers was based on the NCBI Japanese shrimp reference genome (https: / / www.ncbi.nlm.nih.gov / Traces / wgs / JBUDMT01?display=contigs).

[0031] A 20K SNP chip for Japanese shrimp was developed using liquid-phase probe precise localization sequencing and genotyping technology (cGPS). The principle is based on an optimized thermodynamic stability algorithm model to design probes for target region sequences. Synthesized specific probes are used to capture and enrich multiple different target sequences located at different genomic positions through liquid-phase hybridization. Then, sequencing libraries are constructed and high-throughput sequencing is performed on the captured and enriched target genomic sequences to obtain the genotypes of all SNP / InDel sites in the target region.

[0032] Table 1. Location and genotypic information of 20,224 SNP loci in Japanese shrimp. , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , .

[0033] Example 2: The 20K liquid phase chip for Japanese shrimp genotyping The method for genotyping Japanese shrimp samples using the 20K liquid phase chip described in Example 1 specifically includes the following steps: 1. cGPS Library Construction and Quality Control (1) The DNA sample was digested with a fragmentation enzyme, the enzyme ends were repaired, and an A base was added to the 3' end. The fragment size was detected by agarose gel electrophoresis.

[0034] (2) The sequencing adapter and DNA fragment were ligated using T4 ligase, and the ligation product was purified using magnetic beads. The concentration of the purified product was detected by a Qubit fluorescence quantitative PCR instrument, and the fragment size was detected by agarose gel electrophoresis.

[0035] (3) PCR amplification of the purified ligation products: pre-denaturation at 98℃ for 30 s, followed by 6–16 cycles (denaturation at 98℃ for 10 s, annealing at 65℃ for 30 s, extension at 72℃ for 30 s), and a final extension at 72℃ for 5 min. Fragment selection of the amplified products was performed using magnetic beads. The concentration of the selected products was detected using a Qubit quantitative PCR instrument, and the fragment size was detected by agarose gel electrophoresis.

[0036] (4) Take 200 ng of the constructed library, add Japanese shrimp 20K liquid phase chip containing probes and hybridization reagent, and incubate at 50℃ for 16-24 hours to complete the hybridization reaction. Use magnetic beads to capture the target segment, wash the captured product with washing buffer to remove non-specific binding fragments, and then perform a round of PCR amplification. Use a Qubit fluorescence quantitative instrument to detect the library concentration, and agarose gel electrophoresis to detect the fragment size. After the concentration and fragment size are qualified, the cGPS sequencing library construction is complete.

[0037] 2. mGPS Library Construction and Quality Control (1) Primers and multiplex PCR reagents were added to the qualified DNA template to amplify multiple target region sequences simultaneously. The amplified products were purified using magnetic beads. The concentration of the purified products was detected by a Qubit fluorescence quantitative PCR instrument, and the fragment size was detected by agarose gel electrophoresis.

[0038] (2) The adapter sequence required for next-generation sequencing is introduced to both sides of the amplicon product through the second round of PCR reaction. The amplicon product is purified and recovered by magnetic beads. The library concentration is detected by Qubit fluorescence quantitative instrument. The fragment size is detected by agarose gel electrophoresis. After the concentration and fragment size are qualified, the mGPS sequencing library construction is completed.

[0039] 3. Data Analysis The raw data from high-throughput sequencing underwent quality control and filtering. FASTP software was used to remove adapter fragments and low-quality reads, resulting in high-quality Clean Reads. BWA software was used to align the Clean Reads with a reference genome and sort them by position, producing a sorted BAM file. GATK software was used to analyze the sequencing results for variant sites, yielding the original VCF variant result file. The number of supporting reads for different alles at the target site was used to determine the genotype. A homozygous genotype was identified when the proportion of supporting reads for a mutation was ≥0.8 or ≤0.2, and a heterozygous genotype was identified when the proportion was between 0.2 and 0.8. An internally written Perl script was used to process the original VCF variant result file, resulting in a converted VCF variant result file.

[0040] The 20K liquid phase chip for Japanese shrimp provided by this invention can ultimately obtain the genotyping results of each target SNP in a specific individual in a high-throughput manner, thus achieving high-throughput SNP genotyping.

[0041] Example 3: Evaluation of the genotyping effect of the 20K liquid phase chip for Japanese shrimp To verify the genotyping effect of the 20K liquid phase chip for Japanese shrimp, the 20K liquid phase chip for Japanese shrimp designed in Example 1 was used to perform genotyping detection on 96 samples of Japanese shrimp. Four samples were used to set up technical replication experiments, generating a total of 100 sample data.

[0042] Sequencing and data analysis yielded the following results: Figure 2 and Figure 3 As shown, the locus detection rates of all samples ranged from 97.61% to 99.20%, with an average detection rate of 98.33%; the heterozygosity ranged from 31.30% to 35.53%, with an average heterozygosity of 32.81%; and the genotypic consistency rate of duplicate samples was 98.43%. The results indicate that the 20K SNP chip for Japanese shrimp exhibits high target locus detection rate, good stability, and accurate and reliable genotyping results when used for genotyping of the tested materials. It is fully applicable to SNP genotyping detection of different Japanese shrimp samples.

[0043] The 20K SNP chip for Japanese shrimp provided by this invention features high locus polymorphism, abundant functional loci and gene region loci, and uniform distribution of loci on chromosomes. It is a precise and efficient molecular breeding chip. It can be widely used in various application scenarios such as identification of Japanese shrimp germplasm resources and assessment of genetic diversity, construction of genetic maps, QTL mapping, genome-wide association analysis, identification and functional analysis of important trait genes, and genome-wide selection breeding. It is beneficial to improve the breeding efficiency of Japanese shrimp, cultivate new varieties with excellent traits such as fast growth rate and strong disease resistance, and promote the healthy and sustainable development of Japanese shrimp aquaculture.

[0044] Example 4: The 20K liquid phase chip for Japanese shrimp was applied to the discovery and identification of disease resistance genes in Japanese shrimp. Using the genotyping results of the 20K liquid-phase chip for Japanese shrimp obtained in Example 2, the genetic differentiation degree between the two populations (A and B) was detected using VCFtools software. The Fst values ​​at each locus were calculated, and a Manhattan diagram of the locus was plotted. Figure 4 As shown, the first 5% of loci were considered as those with high genetic differentiation (Fst > 0.1160), resulting in 1356 loci. These 1356 loci were annotated using SnpEff. Direct screening using the SnpEff annotations identified 10 candidate genes and variant loci most likely associated with disease resistance. These SNP loci can provide some predictive ability for differences in disease resistance within a population. Association analysis revealed the genetic basis of disease resistance traits in the population, providing a basis for subsequent candidate gene screening and functional validation.

[0045] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions claimed by the present invention.

Claims

1. A probe for detecting SNP molecular marker combinations in Litopenaeus japonicus, characterized in that, The Japanese shrimp SNP molecular marker assemblage consists of 20,224 SNP molecular markers; the 20,224 SNP molecular markers are shown in Table 1, and the location and variation information of the SNP sites are represented in the form of chromosome: physical location: reference genotype / variant allele.

2. A 20K liquid phase chip for Japanese shrimp, characterized in that, The Japanese shrimp 20K liquid phase chip includes probes or primers for detecting Japanese shrimp SNP molecular marker combinations, which consist of 20,224 SNP molecular markers. The 20,224 SNP molecular markers are shown in Table 1. The location and variation information of the SNP sites are represented in the form of chromosome: physical location: reference genotype / variant allele.

3. The application of the probe of claim 1 or the 20K liquid phase chip of Japanese shrimp of claim 2 in molecular marker-assisted breeding of Japanese shrimp.

4. The application of the probe of claim 1 or the 20K liquid phase chip of Japanese shrimp of claim 2 in the genotyping detection of Japanese shrimp.

5. The application according to claim 4, characterized in that, The steps of the genotyping detection include: (1) Extract genomic DNA from the Japanese shrimp samples to be tested; (2) The genomic DNA was digested with a fragmentation enzyme and an A base was added to the 3' end. The sequencing adapter and the DNA fragment were ligated using T4 ligase. The ligation product was purified. The purified ligation product was amplified by PCR. The amplified product was then screened for fragments to construct a library. (3) After adding the Japanese shrimp 20K liquid phase chip and hybridization reagent to the library and incubating, the target segment is captured, and then a round of PCR amplification is performed to construct the cGPS sequencing library. (4) Perform data analysis on the cGPS sequencing library and perform mutation site analysis to obtain the original vcf mutation result file; when the proportion of mutation reads supporting the mutation is ≥0.8 or ≤0.2, the site is judged as homozygous genotype; when the proportion of mutation reads supporting the mutation is between 0.2 and 0.8, the site is judged as heterozygous genotype.

6. The application of the probe of claim 1 or the 20K liquid phase chip of Japanese shrimp of claim 2 in QTL localization analysis of target traits in Japanese shrimp.

7. The application of the probe of claim 1 or the 20K liquid phase chip of Japanese shrimp of claim 2 in genome-wide association analysis of Japanese shrimp.

8. The application of the probe of claim 1 or the 20K liquid phase chip of Japanese shrimp of claim 2 in the discovery or identification of disease resistance genes in Japanese shrimp.

9. The application of the probe of claim 1 or the 20K liquid phase chip of Japanese shrimp of claim 2 in the identification of kinship in Japanese shrimp.

10. The application of the probe of claim 1 or the liquid phase chip of Japanese shrimp of claim 2 in the identification and improvement analysis of Japanese shrimp germplasm resources.

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