A molecular marker related to high temperature tolerance trait of eriocheir sinensis and application thereof

Abstract

The application discloses a molecular marker related to a high-temperature-resistant trait of Eriocheir sinensis and application thereof, relates to the fields of biological breeding technology and molecular marker detection.The nucleotide sequence of the molecular marker is shown in SEQ ID NO.3; and a SNP site exists at the 108th base of the molecular marker, which is a T / C mutation.The application analyzes a gene site related to the high-temperature-resistant trait of Eriocheir sinensis through whole genome association analysis, and filters out a SNP site, namely Chr17_654847, which is significantly associated with the high-temperature-resistant trait.It is found through verification that the TC genotype is an optimal genotype of the Chr17_654847 site, and the high-temperature-resistant trait of an individual with the TC genotype is more prominent.The above-mentioned SNP site can be used as a molecular marker to predict the high-temperature-resistant capability of Eriocheir sinensis, and the detection result is high in accuracy.

Description

Technical Field

[0001] This invention relates to the fields of biological breeding technology and molecular marker detection, and in particular to a molecular marker related to the heat resistance trait of the Chinese mitten crab and its application. Background Technology

[0002] The Chinese mitten crab (Eriocheirsinensis) is an important aquatic economic species. In recent years, extreme high temperatures have become more frequent in summer, and its aquaculture industry has long been constrained by heat stress. Excessively high water temperatures easily lead to metabolic disorders, immunosuppression, and large-scale mortality in crabs, causing significant economic losses. Traditional breeding methods improve heat tolerance through phenotypic selection, but these methods are characterized by long cycles, low efficiency, and poor stability, making it difficult to meet the industry's needs.

[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 heat-resistant 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 heat resistance 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 associated with the heat resistance trait of the Chinese mitten crab and can be used to predict the heat resistance ability of the Chinese mitten crab.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides a molecular marker related to the heat resistance trait of the Chinese mitten crab, the nucleotide sequence of which is shown in SEQ ID NO.3; an SNP site, which is a T / C mutation, is present at the 108th base of the molecular marker.

[0007] Furthermore, the genotype of the SNP site is TT or TC.

[0008] The present invention also provides a primer pair for identifying the heat resistance 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 heat resistance trait of Chinese mitten crab.

[0010] The present invention also provides a kit for identifying the heat resistance trait of Chinese mitten crab, comprising the primer pairs described above.

[0011] The present invention also provides the application of the above-mentioned molecular markers in identifying the heat resistance trait of Chinese mitten crab.

[0012] The present invention also provides the application of the above-mentioned primer pair kit in identifying the heat resistance trait of Chinese mitten crab.

[0013] This invention also provides a method for identifying the heat resistance 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 heat tolerance of the tested Chinese mitten crab was determined: the TC genotype showed higher heat tolerance than the TT genotype.

[0017] Furthermore, the PCR amplification system is as follows: 1 μL of 100 ng / μL template, 0.5 μL of 10 pmol / μL upstream primer, 0.5 μL of 10 pmol / μL downstream primer, 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] This invention uses genome-wide association analysis to identify gene loci associated with the heat tolerance trait of *Eriocheir sinensis* (Chinese mitten crab), screening for a significantly associated SNP locus, Chr17_654847. Validation revealed that the TC genotype is the preferred genotype for this Chr17_654847 locus, with individuals possessing the TC genotype exhibiting more pronounced heat tolerance. Therefore, this SNP locus can be used as a molecular marker to predict the heat tolerance of *Eriocheir sinensis*, with high accuracy. The molecular marker provided by this invention has significant application potential in marker-assisted breeding of heat tolerance in *Eriocheir sinensis*.

[0021] The method for identifying the heat resistance of Chinese mitten crabs in this invention has the advantages of being simple and quick to operate, providing accurate and reliable results, and being inexpensive.

[0022] The molecular markers provided by this invention can be used for early selection and breeding of Chinese mitten crabs with excellent heat resistance, thereby greatly reducing breeding workload, significantly shortening breeding time, accelerating the breeding process, improving breeding efficiency, and reducing breeding costs. This invention has important guiding significance for improving the heat resistance of Chinese mitten crabs, reducing breeding costs, and increasing breeding profits, 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 the heat tolerance trait of Chinese mitten crab;

[0025] Figure 2 QQ plot for GWAS analysis of the heat tolerance trait of Chinese mitten crab;

[0026] Figure 3 A statistical chart showing the heat tolerance of different genotypes of Chinese mitten crabs carrying Chr17_654847; where 1 represents death and 2 represents survival. 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

[0033] Screening of molecular markers related to the heat tolerance trait of Chinese mitten crab:

[0034] 1. Sample Collection

[0035] 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 for a 38°C heat stress experiment. The high-temperature survival data of each crab was measured and recorded. At the same time, tissues of the experimental crabs were collected for genomic DNA extraction and quickly placed in liquid nitrogen. After that, they were transferred to a -80°C freezer for freezing and storage for later use.

[0036] 2. Extraction of genomic DNA

[0037] 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.

[0038] 3. SNP quality control and population structure analysis

[0039] 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.

[0040] 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.

[0041] 4. Genome-wide association analysis of heat resistance trait

[0042] A heat-resistant GWAS was performed using the CMML model in the Genomic Association and Prediction Integration Tool (GAPIT3). Bonferroni correction was applied to determine the genome-wide significance threshold. Manhattan plots and QQ plots were generated using GAPIT3 to evaluate the association results.

[0043] 5. Results Analysis

[0044] After PLINK quality control washing, 401,711 SNP loci were obtained from 335 individuals of *Eriocheir sinensis* for further GWAS analysis. Bonferroni correction was applied to determine the genome-wide significance threshold. GWAS analysis results for the heat tolerance 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 heat tolerance trait of the Chinese mitten crab at the genome level was detected on chromosome 17, namely: Chr17_654847(T>C).

[0045] Example 2

[0046] Validation of molecular markers associated with the heat tolerance trait of the Chinese mitten crab:

[0047] The verification experiment used different populations of Chinese mitten crabs. In addition, 80 experimental crabs were randomly selected to test their heat resistance. The survival data of each crab under heat stress were 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.

[0048] Subsequently, using the extracted DNA as a template, PCR amplification was performed using primers Chr17_654847-F / R to obtain a gene fragment (SEQ ID NO.3) containing the Chr17_654847(T>C) site. The primer sequences are as follows:

[0049] Chr17_654847-F: 5'-GGGCTGTATGTTGCCTAACCA-3' (SEQ ID NO. 1);

[0050] Chr17_654847-R: 5'-TGCCTGCGTACGTACAAGAT-3' (SEQ ID NO. 2).

[0051] The nucleotide sequence of the molecular marker is shown in SEQ ID NO.3. Chr17_654847(T>C) is located at position 108 of SEQ ID NO.3, and the mutation type is T / C.

[0052] SEQ ID NO.3:

[0053] In this context, Y represents T or C; a bold underline indicates a mutation site, and a single underline indicates the positions of the upstream and downstream primers.

[0054] The amplification system used for the PCR reaction was as follows: 1 μL of 100 ng / μL template DNA, 0.5 μL of 10 pmol / μL upstream primer, 0.5 μL of 10 pmol / μL downstream primer, 10 μL of Taq Mix, and double-distilled water to a final volume of 20 μL.

[0055] 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.

[0056] The PCR amplification products were sequenced, and the genotype of each individual was obtained through first-generation sequencing. The heat tolerance phenotype data of individuals with different genotypes were then statistically analyzed. The results are shown in Table 1 and [Table data would be inserted here]. Figure 3 The results showed a significant difference in heat tolerance between the two genotypes of Chinese mitten crab at the Chr17_654847(T>C) locus. The survival rate of individuals with the TT genotype was 4.5%, while that of individuals with the TC genotype reached 41.7%. This demonstrates that this embodiment successfully verified the correlation between this molecular marker and heat tolerance, with the TC genotype showing significantly higher heat tolerance than the TT genotype.

[0057] Table 1. Genotype and trait detection results at the Chr17_654847 (T>C) locus.

[0058]

[0059]

[0060]

[0061] Note: In the survival status under heat stress, 1 represents death and 2 represents survival.

[0062] In summary, the SNP locus (Chr17_654847) screened in this invention is significantly associated with the heat tolerance trait of Chinese mitten crab, and its genotype can be determined by a pair of primers. The operation is simple and reliable, and it has application prospects in molecular marker-assisted breeding and genome selection breeding of Chinese mitten crab.

[0063] 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 heat-resistance trait of *Eriocheir sinensis* in the preparation of a kit for identifying the heat-resistance trait of *Eriocheir sinensis*, characterized in that, 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; The nucleotide sequence of the molecular marker is shown in SEQ ID NO.3; an SNP site exists at the 108th base of the molecular marker, which is a T / C mutation; Individuals with the TC genotype of Chinese mitten crab have a higher tolerance to high temperatures than those with the TT genotype.

2. A kit for identifying the heat resistance trait of the Chinese mitten crab, characterized in that, The kit includes primer pairs for amplifying molecular markers associated with the heat-resistant trait of the Chinese mitten crab; 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; The nucleotide sequence of the molecular marker is shown in SEQ ID NO.3; an SNP site exists at the 108th base of the molecular marker, which is a T / C mutation; Individuals with the TC genotype of Chinese mitten crab have a higher tolerance to high temperatures than those with the TT genotype.

3. The application of a primer pair for amplifying molecular markers associated with the heat-resistance trait of *Eriocheir sinensis* in identifying the heat-resistance trait of *Eriocheir sinensis*, characterized in that... 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; The nucleotide sequence of the molecular marker is shown in SEQ ID NO.3; an SNP site, a T / C mutation, is present at the 108th base of the molecular marker. Individuals with the TC genotype of Chinese mitten crab have a higher tolerance to high temperatures than those with the TT genotype.

4. A method for identifying the heat resistance 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 heat resistance trait of the Chinese mitten crab, to obtain the amplification products of the molecular markers, and then genotyping was performed. 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; The nucleotide sequence of the molecular marker is shown in SEQ ID NO.3; an SNP site exists at the 108th base of the molecular marker, which is a T / C mutation; Based on the genotyping results, the heat tolerance of the tested Chinese mitten crabs was determined: the TC genotype showed higher heat tolerance than the TT genotype.

5. The method according to claim 4, characterized in that, The PCR amplification system consisted of: 1 μL of 100 ng / μL template, 0.5 μL of 10 pmol / μL upstream primer, 0.5 μL of 10 pmol / μL downstream primer, 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: 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.

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