A molecular marker associated with the weight trait of the Chinese mitten crab and its application
By screening out the SNP locus Chr16_16673106G>A associated with the weight trait of Chinese mitten crab, and using PCR amplification to identify the GA genotype, the problem of long breeding cycle was solved, and the effects of improving breeding efficiency and growth traits were achieved.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- FRESHWATER FISHERIES RES INSITUTE OF JIANGSUPROVINCE
- Filing Date
- 2025-09-18
- Publication Date
- 2026-05-26
AI Technical Summary
Existing technologies lack molecular markers for the weight trait of Chinese mitten crab, resulting in long breeding cycles, low efficiency, and an inability to effectively improve its growth traits.
Molecular markers associated with the weight trait of Chinese mitten crab were developed. The GA genotype was identified by PCR amplification using primer pairs through the SNP site Chr16_16673106G>A in the nucleotide sequence, enabling early breeding selection.
It significantly improves breeding efficiency, shortens breeding time, reduces costs, increases growth rate and breeding profits, and is simple to operate with accurate results.
Smart Images
Figure CN120945070B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biotechnology, and in particular to a molecular marker associated with the weight trait of the Chinese mitten crab and its application. Background Technology
[0002] The Chinese mitten crab (Eriocheir sinensis) is an aquaculture species of significant economic value, and genetic improvement of its growth traits is crucial for enhancing aquaculture efficiency. However, the long sexual maturity cycle of the Chinese mitten crab results in an extremely lengthy breeding cycle and low breeding efficiency, severely hindering the development of Chinese mitten crab aquaculture. Therefore, there is an urgent need to develop molecular markers related to growth traits for marker-assisted breeding of the Chinese mitten crab, in order to screen individuals with superior growth traits and apply them to the breeding of the next generation.
[0003] Molecular markers, based on individual differences in the nucleotide sequence of genetic material, directly reflect genetic polymorphism at the DNA level. Among them, single nucleotide polymorphisms (SNPs), as third-generation molecular markers, are numerous and widespread in the genome, thus finding wide application in molecular breeding of plants and animals. The essence of SNPs is DNA sequence polymorphism caused by a single nucleotide variation (substitution, insertion, or deletion) in the genome. The core of marker-assisted breeding lies in using molecular markers closely linked to target trait genes for detection, thereby achieving trait selection. However, currently, there is a lack of molecular markers for marker-assisted breeding of the weight trait in the Chinese mitten crab, which restricts the improvement of this trait. Summary of the Invention
[0004] The purpose of this invention is to provide a molecular marker related to the weight trait of the Chinese mitten crab and its application, in order to solve the problems existing in the prior art. This molecular marker is significantly correlated with the weight trait of the Chinese mitten crab, can be applied to the early breeding of Chinese mitten crabs with excellent growth traits, and has high accuracy.
[0005] To achieve the above objectives, the present invention provides the following solution:
[0006] This invention provides a molecular marker related to the weight trait of the Chinese mitten crab, the nucleotide sequence of which is shown in SEQ ID NO.3; an SNP site, which is a G / A mutation, is present at the 141st base of the molecular marker.
[0007] Furthermore, the genotype of the SNP site is GG or GA.
[0008] The present invention also provides a primer pair for identifying the weight trait of Chinese mitten crab, comprising an upstream primer with a nucleotide sequence as shown in SEQ ID NO.1 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.2.
[0009] The present invention also provides the application of the above-mentioned primer pairs in the preparation of a kit for identifying the weight trait of Chinese mitten crab.
[0010] The present invention also provides a kit for identifying the weight trait of Chinese mitten crab, comprising the primer pair described above.
[0011] The present invention also provides the application of the above-mentioned molecular markers in identifying the weight trait of Chinese mitten crab.
[0012] The present invention also provides the application of the above-mentioned primer pairs or kits in identifying the weight trait of Chinese mitten crab.
[0013] This invention also provides a method for identifying the weight trait of the Chinese mitten crab, comprising the following steps:
[0014] Genomic DNA was extracted from the Chinese mitten crab to be tested;
[0015] Using the genomic DNA as a template, PCR amplification was performed using the primer pairs described above to obtain the amplification products, and then genotyping was performed.
[0016] Based on the genotyping results, the weight trait of the Chinese mitten crabs to be tested was determined: the weight of the GA genotype was significantly higher than that of the GG genotype.
[0017] Furthermore, the PCR amplification reaction system is as follows: 1 μL of 100 ng / μL template DNA, 0.5 μL each of 10 pmol / μL upstream and downstream primers, 10 μL of Taq Mix, and double-distilled water to a final volume of 20 μL.
[0018] Furthermore, the PCR amplification reaction program is as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 35 cycles; 72℃ incubation for 7 min.
[0019] The present invention discloses the following technical effects:
[0020] (1) This invention uses genome-wide association analysis to identify SNP loci associated with the weight trait of *Eriocheir sinensis* (Chinese mitten crab), ultimately screening for candidate SNP loci significantly associated with this trait, namely Chr16_16673106G>A. The results showed that the GA genotype in Chr16_16673106G>A was the preferred genotype. Individuals possessing this genotype exhibited more prominent superior weight traits. Therefore, the aforementioned SNP locus can be used for early selection and breeding of *Eriocheir sinensis* with superior growth traits, with high accuracy. This invention has promising applications in marker-assisted breeding of *Eriocheir sinensis* weight.
[0021] (2) The molecular markers of the present invention can be determined by primers, which has the advantages of simple and fast operation, accurate and reliable results, and low cost.
[0022] (3) The molecular markers of this invention can be used for early selection and breeding of Chinese mitten crabs with excellent weight traits, which can greatly reduce the breeding workload, significantly shorten the breeding time, accelerate the breeding process, improve breeding efficiency, and reduce breeding costs. This has important guiding significance for improving the growth rate of Chinese mitten crabs, reducing the breeding cost of Chinese mitten crabs, and increasing breeding income, and is suitable for widespread application. Attached Figure Description
[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0024] Figure 1 Manhattan plot for GWAS analysis of body weight trait in Chinese mitten crab;
[0025] Figure 2 QQ plot for GWAS analysis of body weight trait in Chinese mitten crab;
[0026] Figure 3 A statistical chart showing the body weight of Chinese mitten crabs carrying different genotypes of Chr16_16673106. Detailed Implementation
[0027] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.
[0028] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.
[0029] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.
[0030] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.
[0031] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.
[0032] Example 1: Screening of molecular markers related to the weight trait of Chinese mitten crab
[0033] 1. Sample Collection
[0034] The Chinese mitten crabs used in this invention came from the breeding base of the Jiangsu River Crab Industry Research Center in China. 335 healthy individuals of Chinese mitten crabs were selected, and the weight data of each crab was measured and recorded. At the same time, tissues of the experimental crabs were collected for the extraction of genomic DNA and quickly placed in liquid nitrogen. After that, they were transferred to a freezer at -80°C for freezing and storage for later use.
[0035] 2. Extraction of genomic DNA
[0036] DNA was extracted from the samples using the TIANGEN kit. DNA content was determined using a UV spectrophotometer, and DNA concentration and purity were determined using 1% agarose gel electrophoresis.
[0037] 3. SNP quality control and population structure analysis
[0038] Whole-genome resequencing was performed using the Illumina HiSeq 2500 platform. SNP data were detected using the Genome Analysis Toolkit (GATK) software. SNPs were quality-controlled filtered according to the following criteria: QD < 2.0, QUAL < 30.0, MQ < 40.0, FS > 60.0, and SOR > 3.0. Subsequently, PLINK was used to filter SNPs based on the criteria of SNP deletion rate < 0.2% and Hardy-Weinberg equilibrium (HWE) > 10. -6 SNPs with minor allele frequencies (MAF) < 0.05 were used for subsequent analysis. Snpeff software was used to annotate the obtained SNPs.
[0039] Before starting the genome-wide association analysis, to avoid the influence of population structure, principal component analysis (PCA) was performed on the quality-controlled data using PLINK software, and then visualized using the R package ggplot2.
[0040] 4. Genome-wide association analysis of body weight trait
[0041] GWAS analysis of body weight data was performed using the CMLM model in the Genome Association and Prediction Integration Tool (TASSEL). Bonferroni correction was applied to determine the genome-wide significance threshold. Manhattan plots and QQ plots were generated using TASSEL to evaluate the association results.
[0042] 5. Results Analysis
[0043] Multiple SNP loci were obtained from 335 individuals of *Eriocheir sinensis* after PLINK quality control washing for further GWAS analysis. Bonferroni correction was applied to determine the genome-wide significance threshold. GWAS analysis results for the body weight trait are shown below. Figure 1 and Figure 2 As shown: Using the Chinese mitten crab (GCF_024679095.1) as the reference genome, a SNP that is significantly associated with the body weight trait of the Chinese mitten crab at the genome level was detected on chromosome 16: Chr16_16673106G>A.
[0044] Example 2: Validation of molecular markers related to the weight trait of the Chinese mitten crab.
[0045] The verification experiment used different populations of Chinese mitten crabs. 100 experimental crabs were randomly selected, and the weight data of each crab was measured and recorded. At the same time, the velvet of each crab was collected and preserved for DNA extraction. The specific procedure for DNA extraction was the same as in Example 1.
[0046] Subsequently, using the extracted DNA as a template, PCR amplification was performed using Chr16_16673106-F / R primers to obtain the gene fragment containing the Chr16_16673106 locus (as shown in SEQ ID NO.3). The primer sequences are as follows:
[0047] Chr16_16673106-F: 5'-TCTCCTCTCCCTCGCCTTTCT-3' (SEQ ID NO.1);
[0048] Chr16_16673106-R: 5'-AGGTGCAGGAATCCTTAGGC-3' (SEQ ID NO. 2).
[0049] The nucleotide sequence of the molecular marker is shown in SEQ ID NO.3. Chr16_16673106 is located at position 141 of SEQ ID NO.3, and the mutation type is G / A.
[0050] SEQ ID NO.3:
[0051] Where R stands for G or A; a bold underline indicates a mutation site; and a single underline indicates the positions of the upstream and downstream primers.
[0052] The reaction system used for PCR was as follows: 1 μL of 100 ng / μL template DNA, 0.5 μL each of 10 pmol / μL upstream and downstream primers, 10 μL of Taq Mix, and double-distilled water to a final volume of 20 μL.
[0053] The PCR reaction program was as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 35 cycles; 72℃ incubation for 7 min.
[0054] The PCR amplification products were sequenced, and the genotype of each individual was obtained through first-generation sequencing. The weight phenotype data of individuals with different genotypes were then analyzed, successfully validating the correlation between this molecular marker and the weight trait. Phenotypic data of individuals carrying the Chr16_16673106G>A locus are shown below. Figure 3 As shown in the results, there were significant differences in body weight between the two genotypes of Chinese mitten crab at the Chr16_16673106G>A locus. Among them, the average body weight of Chinese mitten crabs carrying the GA genotype was significantly higher than that of individuals carrying the GG genotype.
[0055] In summary, the SNP site Chr16_16673106G>A screened in this invention is significantly associated with the weight trait of Chinese mitten crab. Its genotype can be determined by a pair of primers, which is simple and reliable. It has application prospects in molecular marker-assisted breeding and genome selection breeding of Chinese mitten crab.
[0056] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.
Claims
1. The application of a primer pair for amplifying molecular markers associated with the weight trait of *Eriocheir sinensis* in the preparation of a kit for identifying the weight trait of *Eriocheir sinensis*, characterized in that, The nucleotide sequence of the molecular marker is shown in SEQ ID NO.3; an SNP site exists at the 141st base of the molecular marker, which is a G / A mutation; The primer pair includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO.1 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.2; Individuals with the GA genotype of Chinese mitten crab have a significantly higher body weight than those with the GG genotype.
2. The application of a kit for identifying the weight trait of the Chinese mitten crab in identifying the weight trait of the Chinese mitten crab, characterized in that, The kit includes primer pairs for amplifying molecular markers associated with the weight trait of the Chinese mitten crab; The nucleotide sequence of the molecular marker is shown in SEQ ID NO.3; an SNP site, which is a G / A mutation, is present at the 141st base of the molecular marker. The primer pair includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO.1 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.2; Individuals with the GA genotype of Chinese mitten crab had a significantly higher body weight than those with the GG genotype.
3. The application of a primer pair for amplifying molecular markers associated with the weight trait of *Eriocheir sinensis* in identifying the weight trait of *Eriocheir sinensis*, characterized in that... The nucleotide sequence of the molecular marker is shown in SEQ ID NO.3; an SNP site, which is a G / A mutation, is present at the 141st base of the molecular marker. The primer pair includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO.1 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.2; Individuals with the GA genotype of Chinese mitten crab had a significantly higher body weight than those with the GG genotype.
4. A method for identifying the weight trait of the Chinese mitten crab, characterized in that, Includes the following steps: Genomic DNA was extracted from the Chinese mitten crab to be tested; Using the genomic DNA as a template, PCR amplification was performed using primer pairs for amplifying molecular markers related to the weight trait of the Chinese mitten crab, and the amplification products of the molecular markers were obtained and genotyped. The nucleotide sequence of the molecular marker is shown in SEQ ID NO.3; an SNP site, which is a G / A mutation, is present at the 141st base of the molecular marker. The primer pair includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO.1 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.2; Based on the genotyping results, the weight trait of the Chinese mitten crabs to be tested was determined: the weight of the GA genotype was significantly higher than that of the GG genotype.
5. The method according to claim 4, characterized in that, The PCR amplification reaction system was as follows: 1 μL of 100 ng / μL template DNA, 0.5 μL each of 10 pmol / μL upstream and downstream primers, 10 μL of Taq Mix, and double-distilled water to a final volume of 20 μL.
6. The method according to claim 4, characterized in that, The PCR amplification reaction program was as follows: pre-denaturation at 94℃ for 5 min; denaturation at 94℃ for 30 s, annealing at 60℃ for 30 s, extension at 72℃ for 30 s, for a total of 35 cycles; and incubation at 72℃ for 7 min.