Scylla paramamosain trunk weight related SNP (Single Nucleotide Polymorphism) molecular marker, detection primer pair, kit and application

Abstract

The invention discloses a scylla paramamosain trunk weight related SNP (Single Nucleotide Polymorphism) molecular marker, a detection primer pair, a kit and application. The SNP molecular marker is characterized in that the 93rd basic group from the 5'terminal of a nucleotide sequence as shown in SEQ ID NO: 1 is C or T. The SNP molecular marker can predict the trunk weight character of an individual in the early breeding stage, is used for molecular marker-assisted selective breeding (MAS), improves the breeding efficiency and accuracy of scylla paramamosain, shortens the breeding period, reduces the breeding cost and improves the economic benefit.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biotechnology and molecular markers, and relates to a SNP molecular marker related to trunk weight of mud crab, a detection primer pair, a kit and uses. Background Art

[0002] As an important aquaculture product in my country, Scylla paramamosain has become the largest marine aquaculture crab in my country not only because of its high economic value and rapid growth characteristics, but also because of its unique flavor and rich nutritional value. It is deeply loved by consumers. In recent years, with the market's increasing requirements for the quality and traits of blue crabs, the optimization of body shape (especially body weight, of which trunk weight is an important part of body weight, including the weight of the head, thorax and abdomen) has become the focus of breeding work. Trunk weight, as an important economic trait of blue crabs, directly affects the market value and consumer acceptance of blue crabs. In the blue crab farming industry, individuals with larger trunk weight are often more popular in the market, because they not only have more meat, but also have a more spectacular appearance, which can meet consumers' demand for high-quality aquatic products. Therefore, increasing the trunk weight of blue crabs is of great significance to improving the economic benefits of the aquaculture industry, and it is also an important goal in genetic breeding work.

[0003] However, when evaluating traits related to phenotypes, traditional breeding often requires accurate trunk weight data to be obtained after the individual reaches adulthood, which greatly limits the efficiency of early breeding. At the same time, because trunk weight is a complex quantitative trait, it is affected by multiple genes and environmental factors, making it particularly difficult to improve this trait through traditional breeding methods. With the rapid development of high-throughput sequencing technology, genome-wide association study (GWAS) has shown great potential in the genetic analysis of complex traits. This technology has revolutionized the genetic analysis and genetic improvement of important economic traits of farmed animals, from traditional methods that rely on phenotypic selection to the use of molecular markers for assisted selection or whole-genome selection breeding. By performing high-throughput sequencing on the target population of mud crabs and combining phenotypic trait data for association analysis, key genetic loci that affect traits such as trunk weight can be efficiently and accurately located.

[0004] Among the many molecular marker technologies, SNP markers have become the first choice for assisted breeding due to their high abundance, automated detection, low cost and rapidity. Identifying the major quantitative trait loci (QTLs) and key SNP sites that affect the trunk weight of mud crabs is of great significance for accelerating the genetic improvement of the trunk weight trait of mud crabs and improving the economic benefits of aquaculture. Summary of the invention

[0005] The primary purpose of the present invention is to overcome the deficiencies and shortcomings of the prior art and provide a SNP molecular marker located on chromosome 25 of the mud crab and related to body weight.

[0006] To achieve the above object, the present invention provides a SNP molecular marker associated with trunk weight of mud crab, characterized in that the SNP marker is a nucleotide sequence shown in SEQ ID NO: 1, and the 93rd base from the 5' end is C or T. That is, the 11740576th (11740576bp) nucleotide position on chromosome 25 of the mud crab reference genome (National Center for Bioinformation Database retrieval number: GCA_035594125.1) is C>T mutation;

[0007] Furthermore, the trunk weight of individuals with CC genotype and CT genotype of the SNP molecular marker is significantly greater than that of individuals with TT genotype.

[0008] Furthermore, the mud crab includes mud crab and its hybrid varieties or strains.

[0009] The present invention also provides a primer pair for detecting the SNP molecular marker, characterized in that the primer pair has a nucleotide sequence shown in SEQ ID NO: 2-3.

[0010] The present invention also provides a kit for detecting the SNP molecular marker, characterized in that it comprises the primer pair.

[0011] The SNP molecular marker, the primer pair or the kit are used in the breeding of mud crabs and their hybrid varieties or strains.

[0012] The present invention also provides a method for detecting the trunk weight trait of the mud crab, characterized in that the trunk weight trait of the mud crab is determined by detecting the SNP marker on the mud crab to be tested; preferably, the mud crab includes the mud crab and its hybrid varieties or strains.

[0013] Furthermore, the trunk weight trait of the mud crab to be tested is determined by detecting the SNP marker on the mud crab to be tested, including: extracting the genomic DNA of the mud crab to be tested; using the primer pair to perform PCR amplification on the genomic DNA of the mud crab to be tested to obtain a PCR amplification product; sequencing the PCR amplification product to obtain a sequencing result; determining the genotype of the SNP marker of the mud crab to be tested based on the sequencing result; and determining the trunk weight trait of the mud crab to be tested based on the genotype of the SNP molecular marker of the mud crab to be tested.

[0014] Furthermore, the trunk weight of individuals with CC genotype and CT genotype of the SNP molecular marker is significantly greater than that of individuals with TT genotype.

[0015] The present invention also provides a genetic improvement method for mud crabs, characterized in that the method comprises: selecting parent mud crab individuals whose SNP molecular marker genotypes are CC and CT genotypes in the parent mud crab population, and eliminating parent mud crab individuals whose SNP molecular marker genotypes are TT genotypes, so as to increase the frequency of the allele C at the site generation by generation, thereby improving the trunk weight-related traits of the offspring mud crabs; the SNP molecular marker is a nucleotide sequence shown in SEQ ID NO: 1, and the 93rd base from the 5' end is C or T.

[0016] The mud crab described in the present invention includes mud crab and its hybrid varieties or strains.

[0017] The present invention obtains a SNP site significantly correlated with the trunk weight trait of the mud crab through whole genome association analysis. The SNP can be used as a genetic marker and applied to marker-assisted selection, thereby accelerating the genetic improvement process of the trunk weight trait of the mud crab. The present invention verifies the relationship between different SNP allele types and the trunk weight phenotype of the mud crab, confirms the effect of the SNP molecular marker on the trunk weight, and provides a theoretical basis for the breeding application of the SNP marker. Among them, the present invention confirms that the trunk weight of individuals with CC and CT genotypes is significantly greater than that of individuals with TT genotype. The present invention designs a pair of primers that can be used to detect and identify different allele types of the SNP site. The application of this primer pair provides convenience for SNP typing.

[0018] The present invention provides a variety of breeding methods and application schemes based on the SNP molecular marker, including using the SNP to perform genotyping screening on the population; retaining individuals with excellent genotypes for seed preservation; and performing continuous seed selection and matching aimed at increasing the frequency of the target allele. This provides an operational scheme for breeding the trunk weight trait using the SNP molecular marker. The application of the SNP molecular marker can enable the trunk weight trait to obtain a controllable genetic improvement direction, which is conducive to the rapid breeding of new varieties or strains of excellent blue crabs with excellent trunk weight. This will directly increase the commodity value of a single blue crab, which is of great significance to expanding the breeding scale of blue crabs and increasing economic output. The application of the SNP molecular marker will also promote the development of technologies related to molecular breeding and genetic improvement of blue crabs, and will be conducive to promoting the standardization and large-scale breeding of blue crabs. The SNP molecular markers of the present invention and their breeding applications can provide genetic analysis strategies and technical references for molecular breeding of other economic traits. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1This is a Manhattan plot of genome-wide association analysis (GWAS) on the trunk weight trait on chromosome 25 of the mud crab; wherein: the horizontal axis represents the chromosome number of the mud crab; the vertical axis represents the -log10 (P) value. DETAILED DESCRIPTION

[0020] Embodiments of the present invention are described in detail below, and the examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals represent the same or similar elements or elements with the same or similar functions from beginning to end. The embodiments described below by reference to the accompanying drawings are exemplary, are intended to be used to explain the present invention, and are not to be construed as limitations of the present invention. Those who do not indicate specific techniques or conditions in the embodiments are carried out according to the techniques or conditions described in the documents in this area or according to the product specification. Those who do not indicate the manufacturer of reagents or instruments used are all conventional products that can be obtained commercially.

[0021] Example 1: Determination of SNP molecular markers associated with trunk weight traits

[0022] (1) Experimental animals: The samples were 317 mud crabs cultured in Fuzhou, Fujian. The trunk weight phenotype data of each individual was recorded. The muscle tissues of the mud crabs were collected in cryopreservation tubes and stored in a -80°C refrigerator for later use.

[0023] (2) DNA extraction: Genomic DNA was extracted from the muscle tissue of each individual. The Takara genomic extraction kit was used to extract genomic DNA from the samples of the group. The specific operation method was referred to the instruction manual. The DNA quality and concentration were detected by a spectrophotometer, and the DNA samples were stored at -80°C for future use.

[0024] (3) Whole genome resequencing of mud crab:

[0025] DNA samples were sent to Beijing Novogene Technology Co., Ltd., and the whole genome of S. pseudoburrowing was sequenced at 9× depth on the Illumina platform according to the company's standard process of paired-end sequencing. The sequencing data were aligned by BWA software, and the whole genome high-density SNP markers and short fragment insertion / deletion mutations (Indels) were mined by GATK software, and quality control was performed by plink software under the conditions of --mind 0.1--geno 0.1--maf 0.05--hwe 0.000001. The number of SNPs obtained after quality control was 4,042,299.

[0026] (4) Genome-wide association (GWAS) analysis: In order to eliminate the population stratification effect, the present invention uses the linear mixed model single-point regression analysis of GEMMA software to perform GWAS analysis on the above quality-controlled data. The Bonferroni method is used to determine the significance threshold of the degree of association between SNP and trunk weight traits. The genome-level significance threshold is 0.05 divided by the number of effective SNP sites (the number of SNPs after filtering by the plink software indep-pairwise 50 10 0.2 parameter), that is, the genome-level significance threshold is 5.58e-8, that is, 0.05 / 896055 (effective number of SNPs); the chromosome-level significance threshold is 1 divided by the number of effective SNP sites, that is, the chromosome-level significance threshold is 1.12e-6, that is, 1 / 896055 (effective number of SNPs). The results of the GWAS analysis are shown in the accompanying figure. There is a site on chromosome 25 of the mud crab that significantly affects the trunk weight trait, and the strongest associated SNP is the genotype of the C>T mutation at position 11740576 on chromosome 25.

[0027] (5) Correlation analysis between different genotypes and trunk weight traits: Figure 1 It can be seen that the C>T mutation at position 11740576 on chromosome 25 of the molecular marker is extremely significantly correlated with trunk weight-related traits (P<0.001), indicating that this molecular marker can significantly affect the trunk weight-related traits of the mud crab. Therefore, through the auxiliary selection of the C>T mutation at position 11740576 on chromosome 25 of the mud crab SNP site, the trunk weight-related traits of the population can be gradually improved, thereby accelerating the breeding process.

[0028] In addition, according to Table 1, the trunk weight of CC and CT types is larger than that of TT type, indicating that CC and CT are beneficial to these traits. Eliminating the TT genotype of mud crabs to increase the proportion of CC and CT type individuals in the population generation by generation can bring more economic benefits.

[0029] Table 1g. Correlation between 11740576C>T mutation site genotype and trunk weight

[0030]

[0031]

[0032] Example 2: Detailed description of the process of amplifying and sequencing the obtained target DNA sequence

[0033] (1) Design the primer DNA sequence as follows:

[0034] P001-F:5'-ACGCCTGCCTCTTGGATG-3' SEQ ID NO:2;

[0035] P002-R:5'-GTGTGTGTGTGTTACGTTTTAGTC-3' SEQ ID NO:3.

[0036] (2) PCR amplification

[0037] 1 μL DNA template, 10.5 μL double distilled water, 12.5 μL 2×Dream Taq Green PCR, Mastermix (Thermofisher), 0.5 μL primers P001-F and P002-R were added to the 25 μL reaction system. The PCR reaction conditions were: 95°C pre-denaturation for 5 min; 95°C denaturation for 30 s, 60°C annealing for 30 s, 72°C extension for 30 s, 30 cycles; 72°C extension for 10 min.

[0038] (3) DNA sequence determination

[0039] DNA sequence identification: It was carried out at Sangon Biotech Co., Ltd., and the gene fragment was tested for a single positive reaction. The measured sequence was compared with the genome sequence to obtain the mutation of the corresponding SNP site. The sequencing results are shown below:

[0040]

[0041] Note: The Y marked in the sequence table is the mutation site, which is displayed in bold font (the mutated base in brackets is the allele mutation), and the underline at the beginning and end of the sequence indicates the position of the designed primer sequence.

[0042] Example 3: Analysis of the effect of molecular marker SNP site g.11740576C>T

[0043] According to Table 1, the dominant alleles CC and CT of SNP locus g.11740576 can increase the trunk weight by 50.28g and 17.15g respectively compared with the inferior allele TT. Therefore, by molecular marker-assisted selection or genomic selection, the CC and CT types of blue crabs in the population are gradually selected and retained, which can significantly increase the frequency of the dominant allele C, thereby facilitating the weight gain of the blue crab trunk weight trait, accelerating the genetic improvement process of blue crabs, and ultimately effectively improving the economic benefits of blue crab breeding.

[0044] Example 4: Farming Group Verification

[0045] Using the above primer pair P001-F and P002-R, 53 mud crabs (6 months old) from Fuzhou, Fujian were cultured for molecular markers. The SNP site g.11740576C>T was used for typing. Three cases were CC type, 40 cases were CT type, and 10 cases were TT type. The trunk weight of CC type and CT type individuals was 84.12g and 33.82g higher than that of TT type individuals, respectively, and the results were consistent with the actual results.

[0046] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention without departing from the principles and intent of the present invention.

Claims

1. A SNP molecular marker associated with trunk weight in Scylla pseudoburrowing, characterized in that: The SNP molecular marker is a nucleotide sequence shown in SEQ ID NO: 1, wherein the 93rd base from the 5' end is C or T.

2. The SNP marker according to claim 1, characterized in that The trunk weight of individuals with CC genotype and CT genotype of the SNP molecular marker is significantly higher than that of individuals with TT genotype.

3. The SNP molecular marker according to claim 1 or 2, characterized in that: The mud crab includes mud crab and its hybrid varieties or strains.

4. A primer pair for detecting the SNP molecular marker according to any one of claims 1 to 3, characterized in that: The primer pair has the nucleotide sequence shown in SEQ ID NO: 2-3.

5. A kit for detecting the SNP molecular marker according to any one of claims 1 to 3, characterized in that: Comprising the primer pair described in claim 4.

6. Use of the SNP molecular marker according to any one of claims 1 to 3, the primer pair according to claim 4 or the kit according to claim 5 in the breeding of mud crabs and their hybrid varieties or strains.

7. A method for detecting the trunk weight of mud crab, characterized in that: The trunk weight trait of the mud crab is determined by detecting the SNP molecular marker of any one of claims 1 to 3 on the mud crab to be tested; preferably, the mud crab includes the mud crab and its hybrid varieties or strains.

8. The method according to claim 7, characterized in that: include: Extracting genomic DNA of the tested mud crab; Using the primer pair described in claim 4, the extracted genomic DNA is subjected to PCR amplification to obtain a PCR amplification product; Sequencing the PCR amplification product to obtain a sequencing result; Based on the sequencing results, the genotype of the SNP molecular marker of the mud crab to be tested is determined; and based on the genotype of the SNP molecular marker of the mud crab to be tested, the trunk weight trait of the mud crab to be tested is determined.

9. The method according to claim 7, characterized in that: The trunk weight of individuals with CC genotype and CT genotype of the SNP molecular marker is significantly higher than that of individuals with TT genotype.

10. A method for genetic improvement of mud crab, characterized in that: The method comprises: selecting parent mud crab individuals whose SNP molecular marker genotypes are CC and CT genotypes in a parent mud crab population, and eliminating parent mud crab individuals whose SNP molecular marker genotypes are TT genotypes, so as to increase the frequency of the allele C at the site generation by generation, thereby improving the trunk weight-related traits of the offspring mud crabs; the SNP molecular marker is a nucleotide sequence shown in SEQ ID NO: 1, and the 93rd base from the 5' end is C or T.

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