Scylla paramamosain SNP (Single Nucleotide Polymorphism) molecular marker related to third-right-step foot length and section length, detection primer pair, kit and application

Abstract

The invention discloses a scylla paramamosain SNP (Single Nucleotide Polymorphism) molecular marker related to a third-right-step foot length and a section length, a detection primer pair, a kit and application. The SNP molecular marker is characterized in that the 108th basic group from the 5'end of a nucleotide sequence shown as SEQ ID NO: 1 is C or A. The SNP molecular marker can predict the third right step foot length and section length character of an individual in the early breeding stage and is used for molecular marker-assisted selective breeding, the breeding efficiency and accuracy of scylla paramamosain are improved, the breeding period is shortened, the breeding cost is reduced, and the economic benefit is improved.

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 the length of the third right leg segment of a mud crab, a detection primer pair, a kit and uses. Background Art

[0002] Scylla paramamosain is an important product of aquaculture in my country. With its high economic value and fast growth rate, it has become the main species of farmed mud crab in the southeastern coastal areas of my country. In recent years, with the continuous improvement of people's living standards, the awareness of the deliciousness and nutrition of Scylla paramamosain has increased, the scale of Scylla paramamosain farming has gradually expanded, and consumers have also put forward higher requirements for the quality and traits of mud crabs, often hoping that the mud crabs have a larger and better body shape. Among the many biological characteristics of Scylla paramamosain, the length of the third right leg long segment as an important morphological index is not only directly related to the movement and survival competitiveness of Scylla paramamosain, but also proportional to the meat carrying capacity of the long segment, showing important application value in the improvement of mud crab varieties and the selection of high-quality seedlings.

[0003] In traditional breeding, phenotypic data can often only be obtained after the individual reaches adulthood, making it difficult to evaluate and select the individual's phenotype at an early stage. With the development of high-throughput sequencing technology, genome-wide association study (GWAS) based on whole-genome genetic variation has been effective in the genetic analysis of complex traits. The genetic analysis and genetic improvement methods of important economic traits of farmed animals have also evolved from traditional breeding methods through phenotypic selection to breeding methods using molecular markers for marker-assisted selection. By performing high-throughput sequencing on the target population, developing genome-wide molecular markers, and combining phenotypic trait data for genome-wide association analysis, the collected traits can be located and analyzed, which is efficient and practical, and is increasingly widely used in genetic breeding and trait improvement of economic species.

[0004] Among the many molecular marker technologies, single nucleotide polymorphism (SNP) markers have become the first choice for molecular marker-assisted breeding due to their high abundance, automated detection, low cost and rapidity. Identifying the quantitative trait locus (QTL) and key mutation sites that affect the length of the third right leg segment of the mud crab will greatly accelerate the genetic progress of the third right leg segment length trait of the mud crab, thereby improving the economic benefits of the mud crab farming industry. 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 1 of the mud crab and associated with the length of the third right walking leg segment.

[0006] To achieve the above object, the present invention provides a SNP molecular marker associated with the length of the third right leg segment of the mud crab, characterized in that the SNP marker is a nucleotide sequence shown in SEQ ID NO: 1, and the 108th base from the 5' end is C or A. That is, the 40587389th (40587389 bp) nucleotide position C>A mutation on chromosome 1 of the mud crab reference genome (National Center for Bioinformation Database retrieval number: GCF_035594125.1);

[0007] Furthermore, the length of the third right leg segment of individuals with AA genotype and AC genotype of the SNP molecular marker was significantly longer than that of individuals with CC 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 length trait of the third right leg segment of the mud crab, which is characterized in that the length trait of the third right leg segment of the mud crab is determined by detecting the SNP marker on the mud crab to be tested.

[0013] Furthermore, the third right leg segment length 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 third right leg segment length 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 length of the third right leg segment of individuals with AA genotype and AC genotype of the SNP molecular marker was significantly longer than that of individuals with CC 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 genotypes of the SNP molecular marker are AA and AC genotypes in the parent mud crab population, and eliminating parent mud crab individuals whose genotypes of the SNP molecular marker are CC genotypes, so as to increase the frequency of allele A at the site generation by generation, thereby improving the length trait of the third right walking leg segment of the offspring mud crabs, and the SNP molecular marker is a nucleotide sequence shown in SEQ ID NO: 1, and the 108th base from the 5' end is C or A.

[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 associated with the length of the third right leg segment 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 length of the third right leg segment of the mud crab. The present invention verifies the relationship between different SNP allele types and the phenotype of the length of the third right leg segment of the mud crab, confirms the effect of the SNP marker on the length of the third right leg segment, and provides a theoretical basis for the breeding application of the SNP marker. Among them, the present invention confirms that the length of the third right leg segment of individuals with AA and AC genotypes is significantly higher than that of individuals with CC 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 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 third right leg length segment length trait using the SNP marker. The application of the SNP marker can enable the third right leg length segment length 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 longer third right leg length segments. This will directly increase the commodity value of a single blue crab, which is of great significance to expanding the breeding scale of the pseudo-cavern blue crab and increasing economic output. The application of the SNP marker will also promote the development of molecular breeding and genetic improvement-related technologies of the pseudo-cavern blue crab, which is conducive to promoting the standardization and large-scale breeding of the pseudo-cavern blue crab, and improving the overall benefits and competitiveness of the pseudo-cavern blue crab. The SNP marker of the present invention and its breeding application can provide genetic analysis strategies and technical references for molecular breeding of other economic traits. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the Manhattan plot of genome-wide association analysis on chromosome 1 of the mud crab about the length of the third right leg segment; 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 the third right leg length trait

[0022] (1) Experimental animals: The mud crab samples were 356 mud crabs from a cultured population in Fuzhou, Fujian. The phenotypic data of the length of the third right leg segment of each individual were recorded.

[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 the blue crab (National Center for Bioinformation Database Retrieval Number: GCF_035594125.1) was sequenced 9× deep on the Illumina platform according to the company's standard process using the paired-end sequencing method. The sequencing data was aligned by BWA software and GATK software was used to mine high-density SNP markers and short fragment insertion / deletion mutations (Indels) in the whole genome, and the number of SNPs obtained was 45,874,945. The SNPs obtained above were quality controlled using plink software, and the number of SNPs obtained after quality control was 2,051,575.

[0026] (4) Whole genome association (GWAS) analysis: The present invention uses the linear mixed model single point regression analysis of GCTA 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 the length of the third right leg segment. 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 parameters), that is, the genome-level significance threshold is 5.58e-8, that is, 0.05 / 896055 (the number of effective 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 (the number of effective SNPs). The GWAS analysis results are as follows Figure 1 As shown, there is a site on chromosome 1 of the mud crab that significantly affects the length of the third right leg segment. The strongest associated SNP is the C>A mutation at position 40587389 on chromosome 1, with a P value of 2.12e-08.

[0027] (5) Correlation analysis between different genotypes and the length of the third right leg segment: According to Table 1 and Figure 1 It can be seen that the SNP site g.40587389 C>A of the molecular marker is extremely significantly correlated with the length of the left third right leg segment (P<0.001), indicating that this molecular marker significantly affects the length of the left third right leg segment of the mud crab. Therefore, through auxiliary selection of the SNP site g.40587389 C>A of the mud crab, the length of the third right leg segment of the population can be gradually improved, thereby accelerating the breeding process.

[0028] In addition, according to Table 1, the length of the third right leg segment of AA and AC types is longer than that of CC type, indicating that AA and AC are beneficial to the length of the third right leg segment. Eliminating the CC genotype of mud crab can bring more economic benefits. In the process of breeding, we need to eliminate the CC type of mud crab for breeding, so as to increase the proportion of AA and AC type individuals in the population generation by generation.

[0029] Table 1 Correlation between molecular marker SNP site g.40587389C>A and the length of the third right leg segment

[0030]

[0031] Example 2: Amplification and sequencing of target DNA sequences

[0032] (1) Design the primer DNA sequence as follows:

[0033] P001-F: 5'-GCTGCCTGCACATGAAGCTG-3' SEQ ID NO: 2;

[0034] P002-R: 5'-AGGGAGTTTCGAGCAGTGATCG-3' SEQ ID NO: 3.

[0035] (2) PCR amplification

[0036] 2 μL of DNA template was added to the 25 μL reaction system. The DNA template came from 16.5 μL of double distilled water, 2 μL of dNTP, 2.5 μL of 10×Extaq, 0.1 μL of Extaq, and 1 μL each of primers P001-F and P002-R.

[0037] The PCR reaction conditions were as follows: pre-denaturation at 95°C for 5 min; 30 cycles of denaturation at 95°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 30 s; and extension at 72°C 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 M 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 double 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.40587389C>A

[0043] The present invention detects the 108th base mutation site in the SEQ ID NO: 1 sequence, and preliminarily performs an application of the association analysis between its genotype and the third right leg length segment length trait of the pseudo-cavern mud crab, thereby providing a new molecular marker for the molecular marker-assisted selection of the pseudo-cavern mud crab. If the present invention selects all the inferior allele CC types of the SNP site g.40587389C>A that affects the third right leg length segment length trait of the pseudo-cavern mud crab into the superior allele AA and AC types, the third right leg length segment length of the pseudo-cavern mud crab can be increased by 5.54 mm and 3.29 mm, respectively. Therefore, by molecular marker-assisted selection or genomic selection, the AA and AC types of mud crabs in the population are gradually selected and retained, which can significantly increase the frequency of the superior allele A allele, thereby facilitating the growth of the third right leg length segment length trait of the pseudo-cavern mud crab, accelerating the genetic improvement process of the pseudo-cavern mud crab, and ultimately effectively improving the economic benefits of mud crab breeding.

[0044] Example 4: Farming Group Verification

[0045] Using the primer pair P001-F and P002-R, 29 mud crabs (six months old) from Fuzhou, Fujian were genotyped by molecular markers of SNP site g.40587389C>A. 7 cases were TT, 13 cases were TA, and 9 cases were AA. The third right leg segment of TT and TA individuals was 7.06mm and 2.60mm longer than that of CC 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 the length of the third right leg segment of the mud crab, characterized in that: The SNP molecular marker is a nucleotide sequence shown in SEQ ID NO: 1, wherein the 108th base from the 5' end is C or A.

2. The SNP marker according to claim 1, characterized in that The length of the third right leg segment of individuals with AA genotype and AC genotype of the SNP molecular marker was significantly higher than that of individuals with CC 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 length of the third right leg segment of a mud crab, characterized in that: By testing the SNP molecular markers of any one of claims 1 to 3 on the tested mud crab, the length trait of the third right leg segment of the mud crab is determined; 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 result, the genotype of the SNP molecular marker of the tested mud crab is determined; and based on the genotype of the SNP molecular marker of the tested mud crab, the length trait of the third right leg segment of the tested mud crab is determined.

9. The method according to claim 7, characterized in that: The length of the third right leg segment of individuals with AA genotype and AC genotype of the SNP molecular marker was significantly higher than that of individuals with CC genotype.

10. A method for genetic improvement of mud crab, characterized in that: The method comprises: selecting parental mud crab individuals whose genotypes of the SNP molecular marker are AA and AC genotypes in the parental mud crab population, and eliminating parental mud crab individuals whose genotypes of the SNP molecular marker are CC genotypes, so as to increase the frequency of the allele A at the site generation by generation, thereby improving the third right leg length trait of the offspring mud crabs, wherein the SNP molecular marker is a nucleotide sequence shown in SEQ ID NO: 1, and the 108th base from the 5' end is C or A.

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