A snp molecular marker related to body weight of eriocheir sinensis, detection primer pair, kit and use

Abstract

The application discloses a SNP molecular marker related to Scylla paramamosain trunk weight, a detection primer pair, a kit and purposes. The SNP molecular marker is that the 93th base from the 5' end of the nucleotide sequence shown in SEQ ID NO:1 is C or T. The SNP molecular marker can be used for predicting the trunk weight of an individual in early breeding, and is used for molecular marker assisted selection breeding (MAS), 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 trunk weight of Scylla paramamosain, a detection primer pair, a kit and a use. BACKGROUND

[0002] Scylla paramamosain, as an important aquaculture product in China, has become the largest marine crab in China due to its high economic value and rapid growth characteristics, and is also deeply loved by consumers due to its unique flavor and rich nutritional value. In recent years, with the increasing demand for quality and traits of mud crab, especially the optimization of body size (especially body weight, of which trunk weight, including the weight of head and thorax and abdomen, is an important part), has become the focus of breeding work. Trunk weight, as an important economic trait of Scylla paramamosain, directly affects the market value and consumer acceptance of mud crab. In the mud crab breeding 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 the demand of consumers for high-quality aquatic products. Therefore, improving the trunk weight of mud crab is of great significance to improve the economic benefits of the breeding industry and is also an important goal in genetic breeding work.

[0003] However, when evaluating traits related to phenotypes, traditional breeding often needs to obtain accurate trunk weight data after individuals grow up, which greatly limits the efficiency of early selection. At the same time, since 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 way important economic traits of farmed animals are genetically analyzed and genetically improved, from relying on traditional methods of phenotypic selection to using molecular markers for assisted selection or whole-genome selection breeding. By high-throughput sequencing of the target population of Scylla paramamosain and combining phenotypic trait data for association analysis, key genetic loci affecting traits such as trunk weight can be efficiently and accurately located.

[0004] Among numerous molecular marker technologies, SNP markers are the first choice for assisted breeding due to their high abundance, automated detection, low cost, and rapidness. Identifying major quantitative trait loci (QTL) and key SNP sites affecting the trunk weight of Scylla paramamosain is of great significance to accelerate the genetic improvement of the trunk weight trait of Scylla paramamosain and improve the economic benefits of the breeding 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 associated with trunk weight located on chromosome 25 of Scylla paramamosain.

[0006] To achieve the above-mentioned purpose, the present application provides a SNP molecular marker associated with trunk weight of Scylla paramamosain, characterized in that the SNP marker is C or T at the 93nd base from the 5' end of the nucleotide sequence shown in SEQ ID NO: 1. That is, the C>T mutation at the 11740576 (11740576bp) nucleotide site on chromosome 25 of the Scylla paramamosain reference genome (National Biological Information Center database retrieval number: GCA_035594125.1);

[0007] Further, the trunk weight of individuals with CC and CT genotypes of the SNP molecular marker is significantly greater than that of individuals with TT 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 trunk weight trait of Scylla paramamosain, characterized in that the trunk weight trait of the Scylla paramamosain is determined by detecting the SNP marker of the Scylla paramamosain to be tested; preferably, the Scylla paramamosain includes Scylla paramamosain and its hybrid varieties or strains.

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

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

[0015] The application also provides a genetic improvement method of Scylla paramamosain, characterized in that the method comprises the following steps: selecting parent Scylla paramamosain individuals with CC genotype and CT genotype of a SNP molecular marker from a parent Scylla paramamosain population, and eliminating parent Scylla paramamosain individuals with TT genotype of the SNP molecular marker, so as to increase the frequency of allele C at the locus and improve the trunk weight-related traits of the offspring Scylla paramamosain from generation to generation; the SNP molecular marker is a nucleotide sequence shown in SEQ ID NO:1, and the 93th base from the 5' end is C or T.

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

[0017] The application obtains a SNP locus significantly related to the trunk weight trait 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 trunk weight trait of Scylla paramamosain. The application verifies the relationship between different SNP allele genotypes and the trunk weight phenotype of Scylla paramamosain, and proves the influence effect of the SNP molecular marker on the trunk weight, thereby providing a theoretical basis for the breeding application of the SNP marker. In the application, it is proved that the trunk weight of individuals with CC genotype and CT genotype is significantly greater than that of individuals with TT genotype. A pair of primers is designed, which can be used for detecting and identifying different allele types of the SNP locus. 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 molecular marker, including screening of a population by using the SNP for genotyping, keeping of excellent genotype individuals for seed saving, and continuous seed selection and matching for increasing the frequency of a target allele. The application of the SNP molecular marker can make the trunk weight trait obtain a controllable genetic improvement direction, which is beneficial to quickly breed excellent Scylla paramamosain new varieties or strains with excellent trunk weight. This will directly increase the commodity value of a single Scylla paramamosain, and is of great significance for expanding the breeding scale of Scylla paramamosain and improving economic output. The application of the SNP molecular marker will also promote the development of molecular breeding and genetic improvement related technologies of Scylla paramamosain, and is beneficial to promoting the standardized and scaled breeding of Scylla paramamosain. The SNP molecular marker and its breeding application in the application can provide genetic analysis strategies and technical references for the molecular breeding of other economic traits. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1is a Manhattan plot of genome-wide association study (GWAS) on the trunk weight trait on chromosome 25 of Scylla paramamosain; wherein: the abscissa represents the chromosome number of Scylla paramamosain; the ordinate represents the value of -log10(P). DETAILED DESCRIPTION

[0020] Embodiments of the present application are described in detail below with reference to examples illustrated in the accompanying drawings, in which the same or similar components have the same or similar designations throughout the various figures and like reference numerals have been used, where possible, to designate identical or like elements that are common to the figures. The embodiments described below are examples of the present application and are not intended to limit the scope of the present application. Unless otherwise defined, scientific and technical terms used in the examples have the meanings commonly understood by one of ordinary skill in the art. All patents, patent applications, publications, and / or test protocols and procedures are herein incorporated by reference in their entirety.

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

[0022] (1) Experimental animals: 317 samples were from a Scylla paramamosain breeding population in Fuzhou, Fujian. The trunk weight phenotype data of each individual was recorded. The muscle tissue of the above-mentioned Scylla paramamosain was collected in a cryogenic tube and stored in a -80°C refrigerator for standby use.

[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 use.

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

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

[0026] (4) Genome-wide association (GWAS) analysis: In order to eliminate the population stratification effect, the present application uses the linear mixed model single-point regression analysis of GEMMA software to conduct GWAS analysis on the above-mentioned quality-controlled data. The Bonferroni method is used to determine the significance threshold of the association degree of SNPs and trunk weight traits. 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 the figure description. There is a site on chromosome 25 of Scylla paramamosain that significantly affects trunk weight traits, and the most strongly associated SNP is the genotype of C>T mutation at position 11740576 on chromosome 25.

[0027] (5) Association analysis of different genotypes and trunk weight traits: According to the GWAS analysis results, the association of different genotypes and trunk weight traits is analyzed. Figure 1 It can be seen that the C>T mutation at position 11740576 on chromosome 25 of the molecular marker is extremely significantly related to the trunk weight related traits (P<0.001), indicating that this molecular marker can significantly affect the trunk weight related traits of Scylla paramamosain. Therefore, by assisted selection of the SNP site C>T mutation at position 11740576 on chromosome 25 of Scylla paramamosain, 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 traits of CC and CT types are larger than that of TT type, indicating that CC and CT are beneficial to these traits. Eliminating Scylla paramamosain with TT genotype can gradually increase the proportion of CC and CT individuals in the population, which can bring more economic benefits.

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

[0030]

[0031]

[0032] Example 2: Specifically introduces the process of amplification and sequencing of the obtained target DNA sequence

[0033] (1) The primer DNA sequence is as follows:

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

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

[0036] (2) PCR amplification

[0037] In 25 μL of reaction system, 1 μL of DNA template, 10.5 μL of double distilled water, 12.5 μL of 2x Dream Taq Green PCR, Mastermix (Thermofisher), 0.5 μL of primer P001-F and P002-R were added. The PCR reaction conditions were 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.

[0038] (3) DNA sequencing

[0039] DNA sequence sequencing identification: 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: Y in the sequence table is a mutation site, which is displayed in bold font (the mutation base in the bracket, i.e. allele mutation), and the design primer sequence position is displayed in underlined at the beginning and end of the sequence.

[0042] Example 3: Effect analysis of SNP site g.11740576 of molecular marker

[0043] According to Table 1, the SNP site g.11740576 advantage allele genotype CC type and CT type can increase the trunk weight by 50.28 g and 17.15 g, respectively, compared with the disadvantage allele genotype TT type. Therefore, by molecular marker assisted selection or genomic selection, the CC type and CT type of mud crab in the population are gradually selected and reserved, which can significantly increase the frequency of advantage allele C allele, thereby being beneficial to the weight increase of trunk weight traits of mud crab, accelerating the genetic improvement process of mud crab, and finally effectively improving the economic benefit of mud crab breeding.

[0044] Example 4: Verification of breeding population

[0045] The g.11740576C>T SNP site of the 53 cultured T. ferrogamma (6 months old) from Fuzhou, Fujian were genotyped by using the P001-F and P002-R primers. 3 cases were CC type, 40 cases were CT type, and 10 cases were TT type. The trunk weight of the CC type and CT type individuals were 84.12 g and 33.82 g higher than that of the TT type individuals, respectively, which was consistent with the actual results.

[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 of Scylla paramamosain related to trunk weight, characterized in that, The SNP molecular marker is shown in SEQ ID NO: 1, wherein the 93th base from the 5' end is C or T.

2. The SNP molecular marker of claim 1, wherein, 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 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 heavy body trait of Scylla paramamosain, characterized in that, The trunk weight trait 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, Comprise: 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 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.

8. The method of claim 5, wherein, 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.

9. A method of genetic improvement of Scylla paramamosain, characterized by, The method comprises: selecting parent mud crab individuals with CC and CT genotypes of the SNP molecular marker in a parent mud crab population, and eliminating parent mud crab individuals with TT genotype of the SNP molecular marker, so as to increase the frequency of allele C at the site generation by generation, thereby improving the trunk weight-related traits of the offspring mud crab; the SNP molecular marker is shown in SEQ ID NO: 1, wherein the 93th base from the 5' end is C or T.

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