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

Abstract

The application discloses a SNP molecular marker related to body weight and / or body height of Scylla paramamosain, a detection primer pair, a kit and application. The SNP molecular marker is that the 111th 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 body weight and / or body height 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] This invention belongs to the fields of molecular biotechnology and molecular marker technology, and relates to a SNP molecular marker, detection primer pair, kit and application of mud crab related to body weight and / or body height. Background Technology

[0002] The mud crab (Scylla paramamosain), a significant aquaculture product in my country, has become the country's largest farmed marine crab species due to its high economic value and rapid growth. In recent years, with the continuous improvement of people's living standards, there has been a deeper understanding of the deliciousness and nutritional value of mud crabs, leading to an upward trend in their farming scale. At the same time, consumers have also placed higher demands on the quality and characteristics of mud crabs, especially hoping for larger body sizes, particularly optimized weight and height. This has made the weight and height of mud crabs an important goal in breeding efforts.

[0003] In traditional breeding, phenotypic data are often only available after individuals reach adulthood, making it difficult to assess and select individuals for phenotypic development in early stages. With the development of high-throughput sequencing technology, genome-wide association studies (GWAS) have proven highly effective in analyzing complex traits. The methods for genetic analysis and improvement of important economic traits in farmed animals have evolved from traditional phenotypic selection to marker-assisted selection or whole-genome selection. By performing high-throughput sequencing on target populations, developing genome-wide molecular markers, and combining them with phenotypic data for association analysis, all collected traits can be simultaneously analyzed for localization. This method is highly efficient and practical, and its application in the genetic breeding and trait improvement of economically important species is becoming increasingly widespread. To accelerate the genetic improvement of body weight and body height-related traits in the mud crab (Scylla serrata), identifying key mutations affecting body weight and body height using the whole-genome sequence information of the mud crab is crucial.

[0004] Among numerous molecular marker technologies, single nucleotide polymorphism (SNP) marker technology has become the preferred choice for assisted breeding due to its advantages such as high abundance, automated detection, low cost, and rapid detection. Identifying the major quantitative trait loci (QTLs) and key mutation sites affecting the body weight and body height of the mud crab will greatly accelerate the genetic progress of these quantitative traits, thereby improving the economic benefits of mud crab farming. Summary of the Invention

[0005] The primary objective of this invention is to overcome the shortcomings and disadvantages of the prior art and provide an SNP molecular marker located on chromosome 22 of the mud crab that is associated with body weight and / or body height.

[0006] To achieve the above objectives, this invention provides a SNP molecular marker for mud crabs associated with body weight and / or body height, characterized in that the SNP marker is a T or C nucleotide at position 111 from the 5' end of the nucleotide sequence shown in SEQ ID NO: 1. Specifically, it is a C>T mutation at nucleotide position 2237045 (2237045 bp) on chromosome 22 of the mud crab reference genome (National Center for Biotechnology Information Database Retrieval No.: GCA_035594125.1).

[0007] Furthermore, the body weight and / or body height of individuals with the TT and CT genotypes of the SNP molecular markers were significantly higher than those of individuals with the CC genotype.

[0008] Furthermore, the burrowing mud crab includes the burrowing 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 the 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 contains the primer pair.

[0011] The use of the SNP molecular marker, the primer pair, or the kit 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 body height traits of *Scylla scutellarioides*, characterized in that the weight trait of *Scylla scutellarioides* is determined by detecting the SNP markers of the *Scylla scutellarioides* to be tested; preferably, the *Scylla scutellarioides* includes *Scylla scutellarioides* and its hybrid varieties or strains.

[0013] Furthermore, by detecting the SNP marker in the *Scylla serrata* to be tested, the weight and / or body height traits of the *Scylla serrata* to be tested are determined, including: extracting genomic DNA from the *Scylla serrata* to be tested; using the primer pair, performing PCR amplification on the genomic DNA of the *Scylla serrata* to obtain PCR amplification products; sequencing the PCR amplification products to obtain sequencing results; determining the genotype of the SNP marker in the *Scylla serrata* to be tested based on the sequencing results; and determining the weight and / or body height traits of the *Scylla serrata* to be tested based on the genotype of the SNP molecular marker in the *Scylla serrata* to be tested.

[0014] Furthermore, the SNP molecular markers for TT genotype and C T Individuals with the CC genotype had significantly higher body weight and / or height than individuals with the CC genotype.

[0015] This invention also provides a method for genetic improvement of *Scylla scutellaria*, characterized in that the method includes: selecting parental *Scylla scutellaria* individuals with genotypes TT and CT for SNP molecular markers in a parental *Scylla scutellaria* population, and culling parental *Scylla scutellaria* individuals with genotype CC for SNP molecular markers, in order to increase the frequency of the allele T at this locus generation by generation, thereby improving the weight and / or body height-related traits of offspring *Scylla scutellaria*; wherein the SNP molecular marker is the nucleotide sequence shown in SEQ ID NO: 1, with the nucleotide at position 111 (T or C) from the 5' end.

[0016] The mud crabs described in this invention include mud crabs and their hybrid varieties or strains.

[0017] This invention identifies a single Special Nominal (SNP) locus significantly associated with body weight and / or body height traits in the mud crab *Scylla serrata* through genome-wide association analysis. This SNP can serve as a genetic marker for marker-assisted selection, thereby accelerating the genetic improvement of body weight and body height traits in *Scylla serrata*. This invention verifies the relationship between different SNP allele genotypes and the body weight and body height phenotypes of *Scylla serrata*, confirming the influence of this SNP molecular marker on body weight and body height, and providing a theoretical basis for the breeding application of this SNP marker. Specifically, this invention confirms that individuals with the TT and CT genotypes have significantly higher body weight and body height than those with the CC genotype. This invention designs a primer pair that can be used to detect and identify different allele types of this SNP locus. The application of this primer pair facilitates SNP genotyping.

[0018] This invention provides various breeding methods and applications based on this SNP molecular marker, including genotyping and screening of populations using this SNP; retaining individuals with superior genotypes for breeding; and conducting continuous selection and mating aimed at increasing the frequency of target alleles. This provides an operational scheme for breeding body weight and / or body height traits using this SNP molecular marker. The application of this SNP molecular marker allows for controllable genetic improvement of body weight and / or body height traits, which is beneficial for the rapid development of superior new mud crab varieties or strains with excellent body weight and / or body height. This will directly increase the commercial value of individual mud crabs, which is of great significance for expanding the scale of mud crab farming and increasing economic output. The application of this SNP molecular marker will also promote the development of molecular breeding and genetic improvement technologies related to mud crabs, which is conducive to promoting the standardization and large-scale farming of mud crabs. The SNP molecular marker and its breeding applications of this invention can provide genetic analysis strategies and technical references for molecular breeding of other economic traits. Attached Figure Description

[0019] Figure 1 This is a Manhattan plot of genome-wide association analysis (GWAS) on chromosome 22 of the mud crab for the weight trait; where: the horizontal axis represents the chromosome number of the 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 for the body height trait; where: the horizontal axis represents the chromosome number of the mud crab; the vertical axis represents the -log10(P) value. Detailed Implementation

[0021] The embodiments of the present invention are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, 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 obtained from a farmed population of 367 mud crabs in Fuzhou, Fujian Province. Phenotypic data of weight and body height for each individual were recorded. Muscle tissue of the mud crabs was collected and stored in cryovials at -80°C for later use.

[0024] (2) DNA Extraction: Genomic DNA was extracted from the muscle tissue of each individual. The Takara Genomic DNA Extraction Kit was used to extract genomic DNA from the population samples; specific procedures were described in the instruction manual. DNA quality and concentration were measured using a spectrophotometer, and DNA samples were stored at -80°C for later use.

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

[0026] DNA samples were sent to Beijing Novogene Technology Co., Ltd., where the whole genome of *Scylla serrata* was sequenced at 9× depth on the Illumina platform according to the company's standard paired-end sequencing method. Sequencing data were aligned using BWA software, and high-density SNP markers and short insertion / deletion mutations (Indels) were mined using GATK software, yielding 45,874,945 SNPs. The obtained SNPs were then quality controlled using Plink software with the following settings: mind 0.1, geno 0.1, maf 0.05, hwe 0.000001, resulting in 2,051,575 SNPs after quality control.

[0027] (4) Genome-wide association (GWAS) analysis: To eliminate population stratification effects, this invention utilizes linear mixed model single-point regression analysis in GCTA software to perform GWAS analysis on the above-mentioned quality-controlled data. The Bonferroni method was used to determine the significance threshold of the association between SNPs and body weight and height traits. The genomic significance threshold was 0.05 divided by the number of effective SNP loci (the number of SNPs filtered by the indep-pairwise 50 10 0.2 parameter in Plink software), that is, the genomic significance threshold was 5.58e-8, or 0.05 / 896055 (number of effective SNPs); the chromosomal significance threshold was 1 divided by the number of effective SNP loci, that is, the chromosomal significance threshold was 1.12e-6, or 1 / 896055 (number of effective SNPs). The GWAS analysis results are shown in the attached figure. There is a locus on chromosome 22 of the mud crab that significantly affects the traits related to body weight and body height. The strongest associated SNP is the genotype with the C>T mutation at position 2237045 on chromosome 22.

[0028] (5) Association analysis between different genotypes and traits related to body weight and height: Based on Figure 1 and Figure 2 The molecular marker C>T mutation at position 2237045 on chromosome 22 was found to be highly significantly associated with body weight and body height (P<0.001), indicating that this molecular marker can significantly affect the body weight and body height-related traits of *Scylla serrata*. Therefore, by using assisted selection targeting the C>T mutation at position 2237045 on chromosome 22 of *Scylla serrata*, the body weight and body height-related traits of this population can be gradually improved, thereby accelerating the breeding process.

[0029] Furthermore, as shown in Table 1, the TT and CT genotypes exhibit greater and higher body weight and height traits than the CC genotype, indicating that TT and CT are advantageous for these traits. Eliminating CC-type mud crabs to gradually increase the proportion of TT and CT-type individuals in the population can bring greater economic benefits.

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

[0031]

[0032] Example 2: A detailed description of the process of amplifying and sequencing the obtained target DNA sequence.

[0033] (1) The primer DNA sequences are designed 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] To a 25 μL reaction mixture, add 1 μL of DNA template, 10.5 μL of double-distilled water, 12.5 μL of 2×Dream Taq Green PCR kit and Mastermix (Thermofisher), and 0.5 μL each of primers P001-F and P002-R. The PCR reaction conditions were: 225℃ pre-denaturation for 5 min; 30 cycles of denaturation at 225℃ for 30 s, annealing at 60℃ for 30 s, and extension at 72℃ for 30 s; and a final extension at 72℃ for 10 min.

[0038] (3) DNA sequencing

[0039] DNA sequence sequencing identification: Performed at Sangon Biotech Co., Ltd., the gene fragments were sequenced using a forward single reaction. The sequenced data were compared with the genomic sequence to identify mutations at corresponding SNP sites. The sequencing results are shown below:

[0040]

[0041] Note: The Y marked in the sequence listing is the mutation site, displayed in bold (the mutated base in parentheses is the allele mutation). The underline at the beginning and end of the sequence indicates the location of the designed primer sequence.

[0042] Example 3: Analysis of the SNP site g.2237045C>T effect of molecular marker

[0043] Table 1 shows that, compared with the inferior allele CC, the dominant alleles TT and CT at SNP locus g.2237045 increased body weight by 45.98g and 68.74g, respectively; and compared with the inferior allele CC, they increased body height by 3.99mm and 4.04mm, respectively. Therefore, by using marker-assisted selection or genomic selection to gradually retain TT and TC alleles within a population, the frequency of the dominant T allele can be significantly increased, thus promoting weight and height increases in mud crabs, accelerating the genetic improvement process, and ultimately improving the economic benefits of mud crab breeding.

[0044] Example 4: Verification of Farming Population

[0045] Using the aforementioned primer pairs P001-F and P002-R, the SNP site g.2237045C>T was used to genotype 43 *Scylla serrata* (6 months old) farmed in Fuzhou, Fujian Province. One case was genotype TT, 21 cases were genotype CT, and 22 cases were genotype CC. The body weight of TT and CT genotype individuals was 35.33 g and 31.74 g higher than that of CC genotype individuals, respectively, consistent with the observed results. The body height of TT and CT genotype individuals was 5.85 mm and 2.09 mm higher than that of CC genotype individuals, respectively, consistent with the observed results.

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

Claims

1. A SNP molecular marker for mud crabs associated with body weight and / or body height, characterized in that, The SNP molecular marker is shown in SEQ ID NO: 1, where the Y at position 111 from the 5' end is C or T.

2. The SNP molecular marker according to claim 1, characterized in that, Individuals with the TT and CT genotypes of the SNP molecular markers had significantly higher body weight and / or body height than individuals with the CC genotype.

3. The SNP molecular marker according to claim 1 or 2, characterized in that, The term "pseudo-burrowing mud crab" includes pseudo-burrowing mud crab and its hybrid varieties or strains.

4. The use of the SNP molecular markers described in any one of claims 1-3 in the breeding of mud crabs and their hybrid varieties or strains.

5. A method for detecting the weight and / or body height traits of the mud crab *Scylla serrata*, characterized in that, The body weight and / or body height traits of the mud crab were determined by detecting any of the SNP molecular markers described in claims 1-3 on the mud crab to be tested.

6. The method according to claim 5, characterized in that, The term "pseudo-burrowing mud crab" includes pseudo-burrowing mud crab and its hybrid varieties or strains.

7. The method according to claim 5, characterized in that, include: Genomic DNA was extracted from the mud crab to be tested; the extracted genomic DNA was amplified by PCR using primer pairs with the sequences shown in SEQ ID NO:2-3 to obtain PCR amplification products; The PCR amplification products were sequenced to obtain sequencing results; Based on the sequencing results, the genotype of the SNP molecular marker in the tested mud crab is determined; and based on the genotype of the SNP molecular marker in the tested mud crab, the body weight and / or body height traits of the tested mud crab are determined.

8. The method according to claim 7, characterized in that, Individuals with the TT and CT genotypes of the SNP molecular markers had significantly higher body weight and / or body height than individuals with the CC genotype.

9. A method for genetic improvement of the mud crab *Scylla serrata*, characterized in that, The method includes: selecting parental mud crabs with genotypes TT and CT for the SNP molecular marker in a parental mud crab population, and culling parental mud crabs with genotype CC for the SNP molecular marker, in order to increase the frequency of the allele T at this locus generation by generation, thereby increasing the weight and / or body height related traits of offspring mud crabs; the SNP molecular marker is shown in SEQ ID NO: 1, wherein the Y at position 111 from the 5' end is T or C.

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