Scylla paramamosain weight and / or body height related SNP (Single Nucleotide Polymorphism) molecular marker, detection primer pair, kit and application

Abstract

The invention discloses an SNP molecular marker related to body weight and / or body height of scylla paramamosain, a detection primer pair, a kit and application. According to the SNP molecular marker, the 111 basic group from the 5'terminal of a nucleotide sequence shown in SEQ ID NO: 1 is C or T. The SNP molecular marker can predict the body weight and / or body height character of an individual in the early breeding stage and is used for molecular marker assisted selective breeding (MAS), the breeding efficiency and accuracy of 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 invention belongs to the technical field of molecular biotechnology and molecular markers, and relates to a SNP molecular marker related to body weight and / or height 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 due to its high economic value and rapid growth. In recent years, with the continuous improvement of people's living standards, people have a deeper understanding of the deliciousness and nutritional value of Scylla paramamosain, resulting in an upward trend in the scale of Scylla paramamosain farming. At the same time, consumers have also put forward higher requirements for the quality and traits of Scylla paramamosain, especially hoping that Scylla paramamosain has a larger body size, especially the optimization of weight and height, which makes the weight and height of Scylla paramamosain an important goal in breeding work.

[0003] In traditional breeding, phenotype-related measurement data often need to 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) 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 evolved from traditional breeding methods based 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, developing genome-wide molecular markers, and combining phenotypic trait data for association analysis, all collected traits can be located and analyzed at the same time, which is highly efficient and practical, and is increasingly widely used in genetic breeding and trait improvement of economic species. In order to accelerate the genetic improvement of traits related to weight and height of mud crabs, it is crucial to use the whole genome sequence information of mud crabs to identify key mutations that affect the weight and height of mud crabs.

[0004] Among the many molecular marker technologies, single nucleotide polymorphism (SNP) marker technology has become the first choice for assisted breeding due to its high abundance, automated detection, low cost and rapidity. Identifying the major quantitative trait loci (QTLs) and key mutation sites that affect the weight and height of mud crabs will greatly accelerate the genetic progress of the weight and height quantitative traits of mud crabs, thereby improving the economic benefits of mud crab farming. 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 22 of the mud crab and associated with body weight and / or height.

[0006] To achieve the above object, the present invention provides a SNP molecular marker associated with body weight and / or height of mud crab, characterized in that the SNP marker is a nucleotide sequence shown in SEQ ID NO: 1, and the 111th base from the 5' end is T or C. That is, the 2237045th (2237045bp) nucleotide position on chromosome 22 of the mud crab reference genome (National Center for Bioinformation Database retrieval number: GCA_035594125.1) has a C>T mutation;

[0007] Furthermore, the body weight and / or height of individuals with the TT genotype and CT genotype of the SNP molecular marker are significantly higher than those of individuals with the 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 weight and / or height traits of the mud crab, characterized in that the 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 weight and / or height trait of the mud crab to be tested is determined by detecting the SNP marker of 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 weight and / or height 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 TT genotype and C T The body weight and / or height of individuals with the CC genotype were significantly higher than those with the 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 SNP molecular marker genotypes are TT and CT genotypes in the parent mud crab population, and eliminating parent mud crab individuals whose SNP molecular marker genotypes are CC genotypes, so as to increase the frequency of the allele T at the site generation by generation, thereby improving the weight and / or height-related traits of the offspring mud crabs; the SNP molecular marker is a nucleotide sequence shown in SEQ ID NO: 1, and the 111th base from the 5' end is T or C.

[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 weight and / or height traits 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 weight and height traits of the mud crab. The present invention verifies the relationship between different SNP allele types and the weight and height phenotypes of the mud crab, confirms the effect of the SNP molecular marker on weight and height, and provides a theoretical basis for the breeding application of the SNP marker. Among them, the present invention confirms that the weight and height of individuals with TT and CT genotypes are significantly higher than those 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 the 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 weight and / or height traits using the SNP molecular marker. The application of the SNP molecular marker can enable the weight and / or height traits 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 weight and / or height. 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 1 It is a Manhattan plot of genome-wide association analysis (GWAS) on body weight traits on chromosome 22 of mud crab; wherein: the horizontal axis represents the chromosome number of mud crab; the vertical axis represents the -log10 (P) value.

[0020] Figure 2 This is a Manhattan plot of genome-wide association analysis (GWAS) on chromosome 22 of the mud crab about body height; wherein: the horizontal axis represents the chromosome number of the mud crab; the vertical axis represents the -log10 (P) value. DETAILED DESCRIPTION

[0021] 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.

[0022] Example 1: Determination of SNP molecular markers associated with body weight and height traits

[0023] (1) Experimental animals: The samples were 367 mud crabs cultured in Fuzhou, Fujian. The weight and height phenotypic data of each individual were recorded. The muscle tissues of the mud crabs were collected in cryopreservation tubes and stored in a -80°C refrigerator for later use.

[0024] (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.

[0025] (3) Whole genome resequencing of mud crab:

[0026] DNA samples were sent to Beijing Novogene Technology Co., Ltd., and the whole genome of the mud crab 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 GATK software was used to mine high-density SNP markers and short fragment insertion / deletion mutations (Indels) of the whole genome, and the number of SNPs obtained was 45,874,945. The SNPs obtained above were quality controlled using plink software, with the conditions of --mind0.1--geno 0.1--maf 0.05--hwe 0.000001, and the number of SNPs obtained after quality control was 2,051,575.

[0027] (4) Whole genome association (GWAS) analysis: In order to eliminate the population stratification effect, 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 weight and height 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 significance threshold is 5.58e-8, that is, 0.05 / 896055 (effective SNP number); the chromosome level significance threshold is 1 divided by the number of effective SNP sites, that is, the chromosome significance threshold is 1.12e-6, that is, 1 / 896055 (effective SNP number). The results of the GWAS analysis are shown in the accompanying figure. There is a site on chromosome 22 of the mud crab that significantly affects body weight and height related traits. The strongest associated SNP is the C>T mutation genotype at position 2237045 on chromosome 22.

[0028] (5) Analysis of the association between different genotypes and traits related to body weight and height: Figure 1 and Figure 2 It can be seen that the C>T mutation at position 2237045 on chromosome 22 of the molecular marker is extremely significantly correlated with the body weight and height-related traits (P<0.001), indicating that this molecular marker can significantly affect the body weight and height-related traits of the mud crab. Therefore, through the auxiliary selection of the C>T mutation at position 2237045 on chromosome 22 of the mud crab SNP site, the body weight and height-related traits of the population can be gradually improved, thereby accelerating the breeding process.

[0029] In addition, according to Table 1, the weight and height of TT and CT types are larger and higher than those of CC type, indicating that TT and CT are beneficial to these traits. Eliminating the CC genotype of mud crabs to increase the proportion of TT and CT type individuals in the population generation by generation can bring more economic benefits.

[0030] Table 1g. Correlation between genotype of 2237045C>T mutation site and body weight and height

[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'-ATAGTCAATCCCGCTGCTTCTTGC-3' SEQ ID NO:2;

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

[0036] (2) PCR amplification

[0037] 1 μL of DNA template, 10.5 μL of double distilled water, 12.5 μL of 2×Dream Taq Green PCR, Mastermix (Thermofisher), and 0.5 μL of primers P001-F and P002-R were added to the 25 μL reaction system. The PCR reaction conditions were: 225°C pre-denaturation for 5 min; 225°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.2237045C>T

[0043] According to Table 1, the dominant alleles TT and CT of SNP site g.2237045 can increase the weight by 45.98g and 68.74g respectively compared with the inferior allele CC; the dominant alleles TT and CT of SNP site g.2237045 can increase the height by 3.99mm and 4.04mm respectively compared with the inferior allele CC. Therefore, by molecular marker-assisted selection or genomic selection, the TT and TC types of blue crabs in the population are gradually selected and retained, which can significantly increase the frequency of the dominant allele T, thereby facilitating the weight and height increase of blue crabs, 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 pairs P001-F and P002-R, the molecular marker SNP site g.2237045C>T was used to identify 43 mud crabs (6 months old) farmed in Fuzhou, Fujian. One case was TT type, 21 cases were CT type, and 22 cases were CC type. The weight of TT type and CT type individuals was 35.33g and 31.74g higher than that of CC type individuals, respectively, and the results were consistent with the actual results. The height of TT type and CT type individuals was 5.85mm and 2.09mm higher than that of CC 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 body weight and / or height of mud crab, characterized in that: The SNP molecular marker is a nucleotide sequence shown in SEQ ID NO: 1, wherein the 111th base from the 5' end is C or T.

2. The SNP marker according to claim 1, characterized in that The weight and / or height of individuals with the TT genotype and CT genotype of the SNP molecular marker are significantly higher than those with the 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 according to 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 weight and / or height of mud crab, characterized in that: By testing the SNP molecular markers of any one of claims 1 to 3 on the mud crab to be tested, the weight and / or height traits of the mud crab are determined; preferably, the mud crab includes 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 weight and / or height trait of the mud crab to be tested is determined.

9. The method according to claim 7, characterized in that: The weight and / or height of individuals with the TT genotype and CT genotype of the SNP molecular marker are significantly higher than those with the CC 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 TT and CT genotypes in a parent mud crab population, and eliminating parent mud crab individuals whose SNP molecular marker genotypes are CC genotypes, so as to increase the frequency of the allele T at the site generation by generation, thereby improving the weight and / or height-related traits of the offspring mud crabs; the SNP molecular marker is a nucleotide sequence shown in SEQ ID NO: 1, and the 111th base from the 5' end is T or C.

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