A snp molecular marker related to the third right step foot long section length of eriocheir sinensis, a detection primer pair, a kit and a use

Abstract

The application discloses a SNP molecular marker related to the length of the third right step foot of Scylla paramamosain, a detection primer pair, a kit and purposes. 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. The SNP molecular marker can be used for predicting the length of the third right step foot of an individual at an early breeding stage, and can be used for molecular marker assisted selection breeding, so that the breeding efficiency and precision of the Scylla paramamosain are improved, the breeding period is shortened, the breeding cost is reduced, and the economic benefits are improved.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of molecular biology and molecular marker technology, and relates to a SNP molecular marker related to the length of the third right leg of Scylla paramamosain, a detection primer pair, a kit and a use. BACKGROUND

[0002] Scylla paramamosain is an important product of aquaculture in China, which has high economic value and fast growth rate, and has become the main cultivated crab species in the southeast coastal areas of China. In recent years, with the continuous improvement of people's living standards, the recognition of the deliciousness and nutrition of Scylla paramamosain has increased, and the scale of Scylla paramamosain cultivation has gradually expanded. Consumers have higher requirements for the quality and traits of crabs, and often hope that crabs have larger and excellent body size. Among the numerous biological characteristics of Scylla paramamosain, the length of the third right leg as an important morphological index is not only directly related to the movement and survival competition ability of Scylla paramamosain, but also positively correlated with the meat-carrying capacity of the long section, which shows important application value in Scylla paramamosain breeding and high-quality seed selection.

[0003] In traditional breeding, phenotypic data often needs to be obtained after the individual grows up, and it is 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 shown significant effects in the genetic analysis of complex traits. The genetic analysis and genetic improvement method of important economic traits of cultured animals has also developed from traditional breeding methods through phenotypic selection to marker-assisted selection breeding methods using molecular markers. By high-throughput sequencing of the target population, developing molecular markers in the whole genome range, and combining with phenotypic trait data for genome-wide association analysis, the collected traits can be positioned for analysis, which is efficient and practical, and is widely used in genetic breeding, trait improvement and other aspects of economic species.

[0004] Among many molecular marker technologies, single nucleotide polymorphism (SNP) markers have become the first choice for molecular marker-assisted breeding due to their high abundance, automatic detection, low cost and rapidness. Identifying the quantitative trait locus (QTL) and key mutation site affecting the length of the third right leg of Scylla paramamosain will greatly accelerate the genetic progress of the length of the third right leg of Scylla paramamosain, thereby improving the economic benefits of Scylla paramamosain aquaculture. SUMMARY

[0005] The primary purpose of the present application is to overcome the deficiencies and shortcomings of the prior art and provide a SNP molecular marker related to the third right leg foot length of Scylla paramamosain on chromosome 1.

[0006] To achieve the above-mentioned purpose, the present application provides a SNP molecular marker related to the third right leg foot length of Scylla paramamosain, characterized in that the SNP marker is the 108th base from the 5' end of the nucleotide sequence shown in SEQ ID NO: 1 is C or A. That is, the C>A mutation at the 40587389 (40587389bp) nucleotide site on chromosome 1 of the Scylla paramamosain reference genome (National Biological Information Center database retrieval number: GCF_035594125.1);

[0007] Further, the third right leg foot length of individuals with AA and AC genotypes of the SNP molecular marker is significantly higher than that of individuals with CC genotype.

[0008] Further, the Scylla paramamosain includes Scylla paramamosain and its hybrid varieties or strains.

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

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

[0011] The SNP molecular marker, the primer pair or the kit, the use of the SNP molecular marker, the primer pair or the kit in the selection of Scylla paramamosain and its hybrid varieties or strains.

[0012] The present application also provides a method for detecting the third right leg foot length of Scylla paramamosain, characterized in that the third right leg foot length of Scylla paramamosain is determined by detecting the SNP marker of the test Scylla paramamosain.

[0013] Further, the third right leg foot length of Scylla paramamosain is determined by detecting the SNP marker of the test Scylla paramamosain, including: extracting the genomic DNA of the test Scylla paramamosain; using the primer pair, the genomic DNA of the test Scylla paramamosain 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 marker of the test Scylla paramamosain is determined; and based on the genotype of the SNP molecular marker of the test Scylla paramamosain, the third right leg foot length of the test Scylla paramamosain is determined.

[0014] Further, the third right propodus length of the AA genotype and AC genotype individuals of the SNP molecular marker is significantly higher than that of the CC genotype individuals.

[0015] The application also provides a genetic improvement method of Scylla paramamosain, which is characterized by comprising the following steps: selecting parent crab individuals with AA and AC genotypes of the SNP molecular marker in a parent Scylla paramamosain population, eliminating parent crab individuals with CC genotype of the SNP molecular marker, and increasing the frequency of allele A at the site generation by generation, so as to improve the third right propodus length of the offspring Scylla paramamosain, wherein the SNP molecular marker is the nucleotide sequence shown in SEQ ID NO:1, and the 108th base from the 5' end is C or A.

[0016] The Scylla paramamosain in the application includes Scylla paramamosain and its hybrid varieties or strains.

[0017] The application obtains a SNP site significantly related to the third right propodus length of Scylla paramamosain through whole genome association analysis. The SNP can be used as a genetic marker and applied to marker-assisted selection, so as to accelerate the genetic improvement process of the third right propodus length of Scylla paramamosain. The application verifies the relationship between different SNP allele genotypes and the third right propodus length phenotype of Scylla paramamosain, and proves the influence effect of the SNP marker on the third right propodus length, which provides a theoretical basis for the breeding application of the SNP marker. In the application, it is proved that the third right propodus length of the AA and AC genotype individuals is significantly higher than that of the CC genotype individuals. A pair of primers is designed, which can be used to detect and identify different allele types of the SNP site. The application of the primer pair provides convenience for SNP typing.

[0018] The application provides various breeding methods and application schemes based on the SNP marker, including screening of a population by genotyping using the SNP, keeping of excellent genotype individuals for seed saving, and continuous selection of seeds and matching for the purpose of increasing the frequency of a target allele. The application of the SNP marker can make the third right propodus length obtain a controllable genetic improvement direction, which is beneficial to the rapid breeding of new varieties or strains of Scylla paramamosain with longer third right propodus length. This will directly increase the commodity value of a single Scylla paramamosain, and has important significance for expanding the breeding scale of Scylla paramamosain and improving economic output. The application of the SNP marker will also promote the development of molecular breeding and genetic improvement technologies of Scylla paramamosain, and is beneficial to promoting the standardized and scaled breeding of Scylla paramamosain and improving the overall efficiency and competitiveness of Scylla paramamosain. The SNP marker and its breeding application in the application can provide genetic analysis strategies and technical references for molecular breeding of other economic traits. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 Figure 1 is a Manhattan plot of the whole genome association analysis on the third right leg length of the Scylla paramamosain chromosome 1; wherein: the abscissa represents the chromosome number of the Scylla paramamosain; the ordinate represents the -log10(P) value. DETAILED DESCRIPTION

[0020] Embodiments of the present application are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar notations represent the same or similar elements or elements having the same or similar functions throughout. The embodiments described below by reference to the drawings are exemplary and are intended to explain the present application, and are not to be understood as limiting the present application. In the embodiments, specific techniques or conditions are not specified, the techniques or conditions described in the literature in the art or according to the product instructions are used. The reagents or instruments used are not specified by the manufacturer, and are all conventional products that can be obtained commercially.

[0021] Example 1: Determination of SNP molecular markers related to the third right leg length

[0022] (1) Experimental animals: 356 Scylla paramamosain samples from a Scylla paramamosain breeding population in Fuzhou, Fujian. The third right leg length phenotype data of each individual was recorded.

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

[0024] (3) Whole genome resequencing of Scylla paramamosain:

[0025] The DNA samples were sent to Beijing Nuowoziyuan Technology Co., Ltd. According to the company's standard process, the method of paired-end sequencing was used to perform 9x depth sequencing of the whole genome of Scylla paramamosain (National Biological Information Center database retrieval number: GCF_035594125.1) on the Illumina platform. The sequencing data were aligned by BWA software, and the high-density SNP markers and short fragment insertion / deletion mutations (Indels) of the whole genome were mined by GATK software, and the number of SNPs obtained was 45,874,945. The above obtained SNPs were quality controlled by plink software, and the number of SNPs after quality control was 2,051,575.

[0026] (4) Genome-wide association (GWAS) analysis: The present application uses the linear mixed model single-point regression analysis of GCTA software to perform GWAS analysis on the above-mentioned quality-controlled data. The Bonferroni method is used to determine the significance threshold of the degree of association between SNPs and the third right leg length trait. The genome level significant threshold is 0.05 divided by the number of effective SNP sites (the number of SNPs filtered by the plink software indep-pairwise 50 10 0.2 parameters), that is, the genome significant level threshold is 5.58e-8, that is, 0.05 / 896055 (the number of effective SNPs). The chromosome level significant threshold is 1 divided by the number of effective SNP sites, that is, the chromosome significant level threshold is 1.12e-6, that is, 1 / 896055 (the number of effective SNPs). The GWAS analysis results are shown in Table 1 and Figure 1. Figure 1 As shown in Table 1 and Figure 1, there is a significant site affecting the third right leg length on chromosome 1 of the Scylla paramamosain. The most strongly associated SNP is the C>A mutation at position 40587389 on chromosome 1, and the P value is 2.12e-08.

[0027] (5) Association analysis of different genotypes and the third right leg length trait: According to Table 1 and Figure 1 As shown in Table 1 and Figure 1, the SNP site g.40587389 C>A of the molecular marker is extremely significantly related to the left third right leg length (P<0.001), indicating that the molecular marker significantly affects the left third right leg length of Scylla paramamosain. Therefore, the third right leg length of the population can be gradually improved by assisted selection of the SNP site g.40587389 C>A of Scylla paramamosain, thereby accelerating the breeding process.

[0028] In addition, as shown in Table 1, the third right leg length of AA and AC types is longer than that of CC type, indicating that AA and AC are beneficial to the third right leg length. Eliminating Scylla paramamosain with CC genotype can bring more economic benefits. In the breeding process, we need to eliminate Scylla paramamosain with CC genotype to gradually increase the proportion of AA and AC individuals in the population.

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

[0030]

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

[0032] (1) The DNA sequence of the designed primer is as follows:

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

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

[0035] (2) PCR amplification

[0036] 2 μL of DNA template was added in 25 μL of reaction system, and the DNA template was obtained from 16.5 μL of double distilled water, 2 μL of dNTP, 2.5 μL of 10xExtaq, 0.1 μL of Extaq, 1 μL of primer P001-F and 1 μL of primer P002-R.

[0037] The PCR reaction conditions were as follows: 95 °C pre-denaturation for 5 min; 95 °C denaturation for 30 s, 58 °C annealing for 30 s, 72 °C extension for 30 s, 30 cycles; and 72 °C extension for 10 min.

[0038] (3) DNA sequence determination

[0039] DNA sequence determination: performed by Shenguo Bioengineering Co., Ltd., and the gene fragment was measured by a forward single reaction. The measured sequence was compared with the genomic sequence, and the mutation of the corresponding SNP site was obtained. The sequencing results are as follows:

[0040]

[0041] Note: M in the sequence table is a mutation site, which is displayed in bold font (the mutation base in the bracket, i.e., the allele mutation), and the design primer sequence position is displayed as double underlined at the beginning and end of the sequence.

[0042] Example 3: Effect analysis of SNP site g.40587389 C>A of the molecular marker

[0043] The application provides a new molecular marker for molecular marker-assisted selection of Scylla paramamosain by detecting the 108th base mutation site in the sequence of SEQ ID NO: 1, preliminarily performing correlation analysis between the genotype and the third right leg length of Scylla paramamosain, and preliminarily performing correlation analysis between the genotype and the third right leg length of Scylla paramamosain. If the disadvantageous genotype CC of the SNP site g.40587389 C>A affecting the third right leg length of Scylla paramamosain is selected into the advantageous genotypes AA and AC, the third right leg length of Scylla paramamosain can be increased by 5.54 mm and 3.29 mm, respectively. Therefore, by means of molecular marker-assisted selection or genome selection, the AA and AC types of Scylla paramamosain in the population are gradually selected and reserved, the frequency of the advantageous allele A can be significantly increased, the third right leg length of Scylla paramamosain is increased, the genetic improvement process of Scylla paramamosain is accelerated, and finally the economic benefits of Scylla paramamosain breeding are effectively improved.

[0044] Example 4: verification of breeding population

[0045] 29 cultured Scylla paramamosain (six months old) from Fuzhou, Fujian were typed for the SNP site g.40587389C>A using the primer pair P001-F and P002-R. 7 were TT, 13 were TA, and 9 were AA. The length of the third right propodus of the individuals of TT and TA types was increased by 7.06 mm and 2.60 mm respectively compared with that of the individuals of CC type, which was consistent with the actual situation.

[0046] Although the embodiments of the present application have been shown and described above, it should be understood by those skilled in the art that the above embodiments are exemplary and should not be construed as limiting the present application, and those skilled in the art can make changes, modifications, replacements and variations to the above embodiments without departing from the principles and purposes of the present application within the scope of the present application.

Claims

1. A SNP molecular marker associated with the third right pleopod length in Scylla paramamosain, wherein the SNP molecular marker is a SNP marker of a SNP locus, and the SNP locus is located in a region of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-10. The SNP molecular marker is shown as SEQ ID NO: 1, wherein the 108th base from the 5' end is C or A.

2. The SNP molecular marker of claim 1, wherein, The third right step leg length of individuals with AA and AC genotypes of the SNP molecular marker is 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 the mud crab and its hybrid varieties or strains.

4. Use of the SNP molecular marker according to any one of claims 1-3 in the breeding of the mud crab and its hybrid varieties or strains.

5. A method for detecting the long trait of the third right pleopod of Scylla paramamosain, characterized in that, The third right step leg length of the mud crab is determined by detecting the SNP molecular marker according to any one of claims 1-3.

6. The method of claim 5, wherein, The mud crab includes the mud crab and its hybrid varieties or strains.

7. The method of claim 5, wherein, Comprising: extracting the genomic DNA of the mud crab to be tested; performing PCR amplification on the extracted genomic DNA by using the primer pair shown as SEQ ID NO: 2-3, so as to obtain a PCR amplification product; sequencing the PCR amplification product, so as to obtain a sequencing result; determining the genotype of the SNP molecular marker of the mud crab to be tested based on the sequencing result; and determining the third right step leg length of the mud crab to be tested based on the genotype of the SNP molecular marker of the mud crab to be tested.

8. The method of claim 5, wherein, The third right step leg length of individuals with AA and AC genotypes of the SNP molecular marker is significantly higher than that of individuals with CC genotype.

9. A method of genetic improvement of Scylla paramamosain, characterized by, The method comprises: selecting parent mud crab individuals with AA and AC genotypes of the SNP molecular marker in the parent mud crab population, and eliminating parent mud crab individuals with CC genotype of the SNP molecular marker, so as to increase the frequency of allele A at this locus generation by generation, thereby improving the third right step leg length of the offspring mud crab, wherein the SNP molecular marker is shown as SEQ ID NO: 1, wherein the 108th base from the 5' end is C or A.

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