The application discloses a SNP molecular marker related to the back carapace width of eriocheir sinensis on chromosome 2, a detection primer pair, a kit and a use.

Abstract

The application discloses a SNP molecular marker related to Scylla paramamosain back carapace width on chromosome 2, a detection primer pair, a kit and purposes. The SNP molecular marker is that the 202th base from the 5' end of the nucleotide sequence shown in SEQ ID NO:1 is A or T. The SNP molecular marker can be used for predicting the back carapace width of an individual in early breeding, and is 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 present application belongs to the field of molecular biology technology and molecular marker technology, and relates to a SNP molecular marker related to the carapace width of Scylla paramamosain, a detection primer pair, a kit and a use. BACKGROUND

[0002] China's aquaculture industry is undergoing an important stage of pursuing high-quality development. With the improvement of social and economic level, the market demand for the quality of aquatic products is also increasing. Scylla paramamosain belongs to Crustacea, Decapoda, Portunidae and Scylla, and is mainly distributed in Southeast Asia and the southeast coast of China. As one of the main representatives of marine crab culture in China, Scylla paramamosain has rich resources and broad market prospects, and has significant economic value. The carapace width of Scylla paramamosain is considered as one of the morphological characteristics to judge its size specification, and the carapace width is proportional to the size of Scylla paramamosain. Large-sized Scylla paramamosain is more popular with consumers. Therefore, improving the carapace width of Scylla paramamosain is of great significance to improve the economic benefit of Scylla paramamosain, and is also an important measure to adapt to market demand and improve product added value.

[0003] However, traditional breeding methods, especially those relying on artificial selection, face the challenges of high cost, time-consuming and labor-intensive determination, which limits their improvement effect. In order to overcome these challenges and achieve efficient breeding improvement, it is necessary to study the genetic influencing factors of the carapace width of Scylla paramamosain and explore strategies to improve the carapace width using modern breeding techniques. Molecular marker-assisted selection is an advanced genetic improvement technology that can quickly and accurately improve the economic traits of Scylla paramamosain. Among them, single nucleotide polymorphism (SNP) markers have become the preferred technology for genetic marker-assisted breeding due to their high richness and automated detection advantages. By identifying SNP sites related to key economic traits, genetic improvement of these traits can be achieved quickly, providing strong support for the high-quality development of the aquaculture industry.

[0004] With the development of high-throughput sequencing technology, whole-genome resequencing technology has been widely used in the genetic analysis and genetic improvement of important economic traits in agricultural animals. Identifying major quantitative trait loci (QTL) and key mutation sites that affect the carapace width of Scylla paramamosain will greatly accelerate the genetic progress of the carapace width of Scylla paramamosain, thereby improving the economic benefit of Scylla paramamosain aquaculture industry. 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 carapace width of Scylla paramamosain on chromosome 2.

[0006] To achieve the above-mentioned purpose, the present application provides a SNP molecular marker related to the carapace width of Scylla paramamosain, characterized in that the SNP marker is the nucleotide sequence shown in SEQ ID NO: 1, and the 202nd base from the 5' end is A or T. That is, the A>T mutation at the 2785785 (2785785bp) nucleotide site on chromosome 2 of the Scylla paramamosain reference genome (National Biological Information Center database retrieval number: GCF_035594125.1);

[0007] Further, the carapace width of individuals with TT and TA genotypes of the SNP molecular marker is significantly higher than that of individuals with AA 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 sequences 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 are used in the selection of Scylla paramamosain and its hybrid varieties or strains.

[0012] The present application also provides a method for detecting the carapace width of Scylla paramamosain, characterized in that the carapace width of Scylla paramamosain is determined by detecting the SNP marker of the test Scylla paramamosain; preferably, the Scylla paramamosain includes Scylla paramamosain and its hybrid varieties or strains.

[0013] Further, the carapace width 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 to perform PCR amplification on the genomic DNA of the test Scylla paramamosain 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 test Scylla paramamosain based on the sequencing result; and determining the carapace width of the test Scylla paramamosain based on the genotype of the SNP molecular marker of the test Scylla paramamosain.

[0014] Further, the back width of the individuals with TT genotype and TA genotype of the SNP molecular marker is significantly higher than that of the individuals with AA genotype.

[0015] The application further provides a genetic improvement method of Scylla paramamosain, characterized in that the method comprises the following steps: selecting parent crab individuals with TT and TA genotypes of the SNP molecular marker in a parent Scylla paramamosain population, eliminating parent crab individuals with AA genotype of the SNP molecular marker, and increasing the frequency of allele T at the site generation by generation, so as to improve the back width of the offspring Scylla paramamosain, wherein the SNP molecular marker is the nucleotide sequence shown in SEQ ID NO:1, and the 202th base from the 5' end is A or T.

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

[0017] The application is based on whole genome SNP data of Scylla paramamosain, and a SNP site significantly related to the back width is successfully obtained by using whole genome association analysis, which can be used for molecular marker assisted selection to improve the back width, and is expected to accelerate the genetic improvement process of the back width of Scylla paramamosain. Through the method, genetic breeding can be more effectively performed, the economic traits of Scylla paramamosain are improved, and the sustainable development of the Scylla paramamosain breeding industry is promoted.

[0018] The application obtains a SNP site significantly related to the back width of Scylla paramamosain by 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 back width of Scylla paramamosain. The application verifies the relationship between different SNP allele genotypes and the back width of Scylla paramamosain, and confirms the influence effect of the SNP marker on the back width, which provides a theoretical basis for the breeding application of the SNP marker. In the application, it is confirmed that the back width of individuals with TT and AT genotypes is significantly higher than that of individuals with AA genotype. A pair of primers is designed, which can be used for detecting and identifying different allele types of the SNP site. The application of the primer pair provides convenience for SNP typing.

[0019] The present application provides various selection methods and application schemes based on the SNP marker, including screening of populations by genotyping using the SNP; keeping superior genotypic individuals for seed saving; and continuous selection and matching aiming to increase the frequency of the target allele. This provides an operable scheme for selection of the carapace rear edge width trait using the SNP marker. The application of the SNP marker can make the carapace rear edge width trait obtain a controllable genetic improvement direction, which is beneficial to quickly breed a new long carapace rear edge width excellent blue crab variety (strain). This will directly increase the commodity value of a single blue crab, and has important significance for expanding the farming scale of Scylla paramamosain and improving economic output. The application of the SNP marker will also promote the development of Scylla paramamosain molecular breeding and genetic improvement related technologies, and is beneficial to promote the standardized and scaled breeding of Scylla paramamosain. The SNP marker and selection application thereof can provide genetic analysis strategies and technical references for the molecular breeding of other economic traits. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 is a Manhattan plot of whole genome association analysis on the carapace rear edge width trait on chromosome 2 of Scylla paramamosain; wherein: the abscissa represents the chromosome number of Scylla paramamosain; the ordinate represents the -log10(P) value. DETAILED DESCRIPTION

[0021] Embodiments of the present application are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference signs 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 cannot be understood as a limitation of the present application. If specific techniques or conditions are not mentioned in the embodiments, the techniques or conditions described in the literature in the art or according to the product instructions are used. If the reagents or instruments used are not mentioned by the manufacturer, they are all conventional products that can be obtained by purchase.

[0022] Example 1: Determination of SNP molecular marker related to carapace rear edge width trait

[0023] (1) Experimental animals: 367 Scylla paramamosain from a breeding population in Ningde, Fujian were selected. The carapace rear edge width phenotype data of each individual was recorded.

[0024] (2) DNA extraction: Genomic DNA was extracted from the muscle tissue of each individual. Takara genomic extraction kit was used for genomic DNA extraction of the population samples, 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℃ for standby.

[0025] (3) Whole genome resequencing of Scylla paramamosain:

[0026] The DNA sample was sent to Beijing Nuowozhuyuan Technology Co., Ltd. to conduct 9x depth sequencing of the whole genome of Scylla paramamosain on the Illumina platform according to the company's standard process using the paired-end sequencing method. The sequencing data were aligned by BWA software and mined by GATK software to obtain high-density SNP markers and short fragment insertion / deletion mutations (Indels) of the whole genome. 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.

[0027] (4) Whole genome association analysis: The linear mixed model single point regression analysis of GCTA software was used to analyze the above quality controlled data. The Bonferroni method was used to determine the significance threshold of the association degree of SNPs and the back margin width trait. The genome level significant threshold was 0.05 divided by the number of effective SNP sites (the number of SNPs filtered by the indep-pairwise 50, 10, 0.2 parameters of plink software), that is, the genome significant level threshold was 5.58e-8, that is, 0.05 / 896055 (the number of effective SNPs). The chromosome level significant threshold was 1 divided by the number of effective SNP sites, that is, the chromosome significant level threshold was 1.12e-6, that is, 1 / 896055 (the number of effective SNPs). The GWAS analysis results are shown in Table 1 and Figure 1. There is a significant site on chromosome 2 of Scylla paramamosain that affects the back margin width, and the most strongly associated SNP is the A>T mutation at g.2785785 on chromosome 2. Figure 1

[0028] (5) Association analysis of different genotypes and back margin width traits: According to Table 1 and Figure 1 It can be seen that the SNP site g.2785785 A>T of the molecular marker is extremely significantly related to the back margin width trait (P<0.001), which indicates that the molecular marker significantly affects the back margin width trait of Scylla paramamosain. Therefore, the back margin width of the population can be gradually improved by assisted selection of the SNP site g.2785785 A>T of Scylla paramamosain, thereby accelerating the breeding process.

[0029] In addition, according to Table 1, the back margin width of the TT type and the TA type is longer than that of the AA type, indicating that TT and TA are beneficial to the back margin width. Eliminating Scylla paramamosain with AA genotype can bring more economic benefits. In the breeding process, we need to eliminate Scylla paramamosain with AA genotype to gradually increase the proportion of TT and TA individuals in the population.

[0030] Table 1 Correlation of SNP mutation site g.2785785 A>T of the molecular marker and the back margin width trait ​

[0031]

[0032]

[0033] Example 2: Amplification and sequencing of target DNA sequence

[0034] (1) The primer DNA sequence is as follows:

[0035] P001-F: 5'-CGCCACACTTGTCCCTTGTTTT-3' SEQ ID NO: 2;

[0036] P002-R: 5'-ACGCGCACACACACTACCAT-3' SEQ ID NO: 3.

[0037] (2) PCR amplification

[0038] In the reaction system of 25 μL, add 1 μL of DNA template, 10.5 μL of double distilled water, 12.5 μL of 2x Dream Taq Green PCR Mastermix (Thermofisher), and 0.5 μL of primer P001-F and P002-R respectively. The PCR reaction conditions are as follows: 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.

[0039] (3) DNA sequence determination

[0040] DNA sequence determination: performed by Novogene Bioinformatics Technology Co., Ltd., and the forward single reaction of the gene fragment was determined. 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:

[0041]

[0042] Note: W marked in the sequence table is the mutation site, which is displayed with bold font (the mutation base in the parentheses, i.e. allele mutation), and the design primer sequence position is displayed with underlined at the beginning and end of the sequence.

[0043] Example 3: Effect analysis of SNP site g.2785785A>T of molecular marker

[0044] The application provides a new molecular marker for molecular marker assisted selection of Scylla paramamosain by detecting the 202th base mutation site in the sequence of SEQ ID NO: 1, preliminarily performing correlation analysis between genotypes and the trait of the back width of carapace of Scylla paramamosain, and selecting all AA and TA type individuals of the molecular marker affecting the trait of the back width of carapace of Scylla paramamosain into TT type individuals. Only the back width of carapace of Scylla paramamosain can be increased by 3.06 mm and 1.09 mm, and since the back width of carapace of Scylla paramamosain and weight are positively correlated, the weight of Scylla paramamosain can be increased by increasing the back width of carapace of Scylla paramamosain. Therefore, increasing the back width of carapace of Scylla paramamosain provides great potential for the aquaculture industry of Scylla paramamosain. In the SNP molecular marker individual, the economic benefits of Scylla paramamosain can be ultimately improved by selecting the superior allele (T) of the SNP of Scylla paramamosain, thereby increasing the income of aquaculture.

[0045] Example 4: verification of a breeding population

[0046] 29 Scylla paramamosain (six months old) bred in Fuzhou, Fujian are typed by the SNP site g.2785785A>T of the molecular marker by using the above P001-F and P002-R primers. 7 cases are TT, 13 cases are TA type, and 9 cases are AA type. The back width of carapace of the TT type and TA type individuals is increased by 8.37 mm and 1.25 mm, respectively, compared with the back width of carapace of the AA type individual, and the result is consistent with the actual result.

[0047] Although the embodiments of the application have been shown and described above, it should be understood that the above embodiments are exemplary and should not be construed as limiting the 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 application.

Claims

1. A SNP molecular marker located on chromosome 2 and related to the back carapace width of Eriocheir sinensis, characterized in that, The SNP molecular marker is shown as SEQ ID NO: 1, wherein the 202th base from the 5' end is A or T.

2. The SNP molecular marker of claim 1, wherein, The back width of individuals with TT genotype and TA genotype of the SNP molecular marker is significantly higher than that of individuals with AA 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 trait of wide posterior carapace margin in Scylla paramamosain, characterized in that, The back width 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 of 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 back width 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 back width of individuals with TT genotype and TA genotype of the SNP molecular marker is significantly higher than that of individuals with AA genotype.

9. A method of genetic improvement of Scylla paramamosain, characterized by, The method comprises: selecting parent crab individuals with TT and TA genotypes of the SNP molecular marker in the parent mud crab population, eliminating parent crab individuals with AA genotype of the SNP molecular marker, and increasing the frequency of allele T at this site generation by generation, so as to improve the back width related traits of the offspring mud crab, wherein the SNP molecular marker is shown as SEQ ID NO: 1, wherein the 202th base from the 5' end is A or T.

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