Molecular marker S617 for identifying ostrea rivularis glycogen content character related gene CLASRP and application of molecular marker S617

Abstract

The invention provides a molecular marker S617 for identifying ostrea rivularis glycogen content character related gene CLASRP and application of the molecular marker S617, and belongs to the technical field of molecular marker breeding. The nucleotide sequence of the molecular marker S617 is as shown in SEQ ID NO.1, and the genotype CC of the 466th base of the molecular marker S617 is a genotype with high glycogen content. The invention also provides a primer for detecting the molecular marker S617, the nucleotide sequences of an amplification primer are shown as SEQ ID No.2 and SEQ ID No.3, and the sequence of an extension primer is shown as SEQ ID No.4. The molecular marker S617 can be used for increasing the breeding speed of the high-glycogen-content character variety of the ostrea rivularis, a large amount of manpower and material resources are saved, the progress of improved variety breeding is promoted, healthy and sustainable development of the ostrea rivularis industry is facilitated, and the molecular marker S617 has a wide market application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular marker breeding, and specifically relates to a molecular marker S617 for identifying a gene CLASRP related to a glycogen content trait of an Omi oyster and an application thereof. Background Art

[0002] Omi oyster ( Crassostrea ariakensis ) is an estuarine species of the genus Crassostrea, with biological characteristics such as a wide range of temperature and salt adaptability, large individuals, and fast growth rate. In the south, the Omi oyster is the main aquaculture variety in Guangdong and Fujian provinces. Due to its significant growth advantages, it has gradually become one of the emerging commercial economic species in the local area in recent years. Due to its significant ecological and economic effects, the scale of aquaculture of Omi oysters in the north is getting larger and larger, and breeding work is also deepening. With the continuous expansion of market demand for high-end oysters and the improvement of people's living standards, high-quality oysters with sweet taste are deeply loved by consumers. Shandong Omi oysters are mostly monomers and large individuals, with a sweet taste, and are recognized by the high-end market. Studies have found that the level of glycogen content directly affects the sweet flavor of oysters, and is often used as an important criterion for judging the meat quality traits of oysters. Therefore, work on improving the quality traits of high-glycogen Omi oysters is urgently needed.

[0003] Molecular marker-assisted breeding has been widely used in the field of aquatic breeding. Single nucleotide polymorphisms (SNPs) are the most abundant sequence variation category in the genome, with the characteristics of high density, representativeness and genetic stability. The use of this molecular marker plays an important role in pre-selecting excellent traits of aquatic animals, shortening the breeding cycle, improving breeding efficiency, and promoting the creation of new germplasm of aquatic animals and high-quality development of the industry. At present, there are very few reports on molecular markers for the high glycogen content trait of Omi oysters. Therefore, the development of molecular markers related to the high glycogen content trait is of great significance to the genetic selection of Omi oysters. Summary of the invention

[0004] The purpose of the present invention is to provide a molecular marker S617 for identifying the gene CLASRP related to the glycogen content trait of the Omi oyster and its application. The present invention uses the polymorphic site filtering and comparison analysis of sequencing data and the PCR sequencing method, and then gradually screens and verifies the markers to finally obtain a new molecular marker S617 related to the glycogen content trait of the Omi oyster. The molecular marker S617 can accurately and efficiently identify the varieties of the Omi oyster with high glycogen content traits, which is conducive to the screening and breeding of the glycogen content trait of the Omi oyster.

[0005] In order to achieve the above-mentioned invention objectives, the present invention adopts the following technical solutions: The invention provides a molecular marker S617 for identifying a gene CLASRP related to a glycogen content trait of an Omi oyster. The nucleotide sequence of the molecular marker S617 is shown in SEQ ID No.1.

[0006] Furthermore, the molecular marker S617 is located on chromosome 6, and the 466th base in the nucleotide sequence of the molecular marker S617 is T or C.

[0007] Furthermore, the genotype CC of the 466th base of the molecular marker S617 is a high glycogen content trait genotype.

[0008] Furthermore, the molecular marker S617 is a SNP marker.

[0009] The present invention also provides amplification primers and extension primers for the molecular marker S617. The nucleotide sequences of the amplification primers are shown in SEQ ID No.2 and SEQ ID No.3, and the extension primer sequence is shown in SEQ ID No.4.

[0010] The present invention also provides the use of the molecular marker S617 or the amplification primer and the extension primer in breeding the Omi oyster variety with the trait of high glycogen content.

[0011] Furthermore, the screening step is: the step of breeding the Omi oyster variety with a high glycogen content trait is: extracting the Omi oyster sample DNA as a template, using the amplification primers S617 upstream primer and S617 downstream primer of the molecular marker S617 for PCR amplification, treating the obtained amplicon with alkaline phosphatase to remove dNTPs, further performing an extension reaction and desalting, and using the desalted product for mass spectrometry detection. If the genotype of the 466th base of the molecular marker S617 in the test result is CC, then the Omi oyster sample to be tested is an Omi oyster variety with a high glycogen content.

[0012] Furthermore, the conditions for the PCR amplification are: pre-denaturation at 94°C for 3 min, denaturation at 94°C for 30 s, annealing at 56°C for 25 s, and extension at 72°C for 30 s, for a total of 40 cycles.

[0013] Furthermore, the conditions of the extension reaction are: pre-denaturation at 94°C for 30 s, denaturation at 94°C for 5 s, annealing and extension for 5 cycles including annealing at 52°C for 5 s and extension at 80°C for 5 s, for a total of 40 cycles.

[0014] The present invention also provides a kit for breeding a variety of Crassostrea gigas with high glycogen content, wherein the kit contains the molecular marker S617 and / or the amplification primer and the extension primer.

[0015] The present invention also provides the application of the molecular marker S617 in genetic diversity analysis, germplasm identification and genetic map construction of Omi oysters.

[0016] Compared with the prior art, the present invention has the following advantages and beneficial effects: The present invention provides a SNP molecular marker site S617 of the glycogen content trait-related gene CLASRP by sequencing and analyzing the whole genome of the Omi oyster. Compared with the determination of glycogen content by biochemical reagents, near infrared and other methods, this method identifies and screens individuals of Omi oysters with high glycogen content at the molecular level, is simple and easy to operate, and can accurately and quickly screen target individuals; it can also test the parental genotype through non-destructive operation, and guide the selection of parents according to the high glycogen content genotype, thereby shortening the breeding process of high glycogen Omi oysters, and providing a reliable technical means for the quality breeding of Omi oysters.

[0017] The identification method of the present invention has strong applicability. The oyster DNA extraction method, genome resequencing and PCR amplification all adopt conventional methods in the art. The method is simple and efficient, and the detection is low-cost. It has wide applicability in the field of screening individuals of Omi oysters with high glycogen content traits. Therefore, the molecular marker S617 and the primer pair and identification method used to identify the molecular marker have broad application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is the mass spectrometry result diagram of three genotypes of the molecular marker S617. DETAILED DESCRIPTION

[0019] The technical solution of this invention is further described in detail below in conjunction with specific embodiments.

[0020] The Omi oysters used in the present invention were taken from five different geographical groups, namely Binzhou, Shandong, Dongying, Shandong, Luchao Island, Shanghai, Guangzhou, Guangdong, and Beihai, Guangxi, with a total of 250 individuals. Six tissues, including gonads, hepatopancreas, mantle, lip, gills, and adductor muscle, were taken for the determination of glycogen content. At the same time, adductor muscle tissue was taken and preserved in 95% alcohol for DNA extraction.

[0021] Example 1 1. Screening of molecular markers related to glycogen content 1. Library construction, sequencing and data analysis QIAamp DNA kit was used to extract DNA from adductor muscle tissue of Crassostrea ovalis. The specific steps were as follows: (1) Place 10 mg of tissue sample in a 1.5 mL centrifuge tube and add 180 μL of Buffer ATL. Equilibrate to room temperature (15–25°C).

[0022] (2) Add 20 μL of proteinase K, vortex to mix for 15 seconds, and incubate in a thermomixer at 56°C overnight until the sample is completely lysed.

[0023] (3) Add 200 μL of Buffer AL and vortex to mix for 15 seconds.

[0024] (4) Add 200 μL of ethanol, vortex to mix for 15 seconds, and incubate at room temperature for 5 minutes.

[0025] (5) Centrifuge briefly and transfer the supernatant to a QIAamp MinElute column. Place the QIAamp MinElute column in a clean 2 mL collection tube and centrifuge at 6000 × g for 1 min at room temperature. Discard the collection tube containing the flow-through.

[0026] (6) Add 500 μL of Buffer AW1 to the QIAamp MinElute column and centrifuge at 6000 × g for 1 min at room temperature. Discard the collection tube containing the flow-through.

[0027] (7) Repeat step (6).

[0028] (8) Add 700 μL of ethanol to the QIAamp MinElute column and centrifuge at 6000 × g for 1 min at room temperature. Discard the collection tube containing the flow-through and centrifuge at 20,000 × g for 3 min at room temperature to completely dry the membrane.

[0029] (9) Place the QIAamp MinElute column in a clean 1.5 mL centrifuge tube. Carefully open the column cap and incubate at room temperature for 10 minutes.

[0030] (10) Add 30 μL of Buffer ATE to the center of the membrane, cover the lid, incubate at room temperature for 1 minute, and centrifuge at 20,000 × g for 1 minute at room temperature. The liquid in the centrifuge tube is the DNA sample.

[0031] (11) Agarose gel electrophoresis was used to detect the integrity of DNA and whether it contained protein and RNA contamination. The purity and concentration of DNA were detected using an ultra-micro spectrophotometer. The DNA was accurately quantified using Qubit 3.0.

[0032] High-quality DNA was extracted from the muscle for library construction and short-read sequencing. A library with a total length of approximately 400 bp was constructed, and the library was sequenced using the BGISEQ-500 platform to obtain Raw reads. Reads containing adapters and reads with an N ratio greater than 10% and low-quality reads were removed to obtain Clean reads for subsequent data analysis.

[0033] 2. Marker Detection and Annotation The clean reads were compared with the reference genome using Burrows-wheeler aligner (BWA) software, and format conversion and sorting were performed using Samtools software. The Genome analysis toolkit (GATK) software was used to call SNPs, annotate SNP sites, determine the variant sites and corresponding gene information, and obtain a total of 1,918,741 high-quality SNPs for subsequent genome-wide association (GWAS) analysis.

[0034] 3. Glycogen content determination The modified anthrone method was used to detect the glycogen content in six different tissues of 250 Crassostrea gigas. The specific steps are as follows: (1) Freeze-dry the fresh tissue for 48 h, grind it into powder (20 ± 1 mg) under freezing conditions and transfer it to a 10 mL centrifuge tube; (2) Add 2 mL of 30% potassium hydroxide solution (w / w) and digest in a water bath for 45 min; (3) Adjust the total volume to 5 mL with distilled water, centrifuge at 4000 × g for 8 min at room temperature, and collect the supernatant; (4) Dilute 100 μL of the supernatant 10-fold and mix with 200 μL of 0.2% (w / v) anthrone sulfuric acid solution on ice. (5) Place the mixture in a water bath for 10 min, cool it with tap water, and let it stand at room temperature for 30 min. Take 200 μL of the mixture and measure the optical density (OD) at 620 nm using an ELISA reader. (6) Use a glucose solution with a concentration of 0.01 to 0.2 mg / mL to establish a standard curve, and calculate the glycogen content according to the formula: C = C0 × (5 mL / W) × 10 × 0.9, where C is the glycogen content of the sample (mg / g), C0 is the glucose content in the test solution calculated according to the standard curve (mg / mL), W is the weight of the sample to be tested (g), 5 mL is the fixed volume, 10 is the dilution factor, and 0.9 is the conversion factor of glucose to glycogen.

[0035] 1. GWAS analysis The glycogen content data of six different tissues were reduced in dimension using principal component analysis (PCA), and the first principal component (PC1) was used as phenotypic data for GWAS analysis. GWAS analysis was performed based on a mixed linear model (MLM) using EMMAX software to explore SNPs associated with high glycogen content. In order to reduce false positive associations, Vcftools software was used to perform quality control on SNPs, with the following parameters: "--max missing 0.99--maf 0.05--minDP 4--minGQ 10--minQ30--min meanDP 10". In order to screen as many candidate genes related to glycogen content as possible, the threshold for GWAS analysis was set to P < 1×10 -5 Finally, 132 SNP sites significantly associated with glycogen content were obtained ( P < 2.61×10 -8 ),according to P The values ​​were sorted from small to large, and the first 10 SNP sites were selected for verification.

[0036] 2. Validation of molecular markers related to high glycogen content 100 oysters cultured in this study were randomly selected for growth trait measurement and glycogen content determination by near-infrared spectroscopy. The oysters were arranged from high to low according to glycogen content. The first 20 individuals were selected as the high glycogen group and the last 20 individuals were selected as the low glycogen group. The glycogen contents are shown in Table 1.

[0037] Table 1 Glycogen content

[0038] The above 40 individuals were used to detect the 10 SNP sites screened by GWAS. First, the PCR amplification primers and single base extension primers of the test sites were designed using GenotypingTools and Massarray Assay Design software. The design conditions were: primer length 18~30 bp, PCR product 80-200bp; Tm value 55~65°C, annealing temperature around 60°C; GC content 40~70%. According to the above DNA extraction method, DNA of the high glycogen group and the low glycogen group were obtained, and PCR amplification was performed using this as a template.

[0039] The amplification system was as follows: 0.1 μL dNTP, 0.4 μL MgCl2, 0.1 μL HotStar Taq (5 U / μl), 0.5 μL PCR buffer, 0.25 μL each of upstream and downstream primers (10 μmol / L), 1 μL DNA, 2.4 μL water. The PCR amplification conditions were: 94°C pre-denaturation for 3 min, 94°C denaturation for 30 s, 56°C annealing for 25 s, 72°C extension for 30 s, for a total of 40 cycles.

[0040] After the PCR reaction, the PCR product was treated with SAP (shrimp alkaline sulfatase) to remove dNTPs in the reaction. The reaction system was: 0.17 μL SAP buffer (10X), 0.3 μL SAP enzyme (1.7U / μL), and 1.53 μL water. The extension reaction was performed according to the following procedure: 94 °C pre-denaturation for 30 s, 94 °C denaturation for 5 s, annealing and extension for 5 cycles including 52 °C annealing for 5 s and 80 °C extension for 5 s, for a total of 40 cycles. Reaction system: 2 μL EXTEND Mix, 7 μL SAP+PCR reactant. After desalting, the extension reaction product was subjected to mass spectrometry SNP typing technology for SNP site detection. SPSS 26.0 software was used to perform a chi-square test to test the genotype frequencies of the differential SNP sites between the high glycogen group and the low glycogen group. P < 0.01 was considered to be a very significant difference. P A value < 0.05 was considered to be significantly different, otherwise it was considered to be non-significant.

[0041] According to Table 2 and Figure 1 As shown, the mutation type of the 466th base site of the molecular marker S617 (40518787th base site of chromosome 6) is T / C, and there are three genotypes, among which the genotype CC accounts for 65% in the high glycogen group, and the genotype TT accounts for 85% in the low glycogen group (Table 2). There is a very significant difference between the high glycogen group and the low glycogen group at this site ( P <0.01), indicating that the marker was extremely significantly correlated with the glycogen content trait. Therefore, the genotype CC at this site was considered to be a high glycogen genotype.

[0042] It was concluded that the molecular marker for regulating glycogen content in Omi oysters is S617. The nucleotide sequence of the molecular marker S617 is shown in SEQ ID No. 1, and a partial sequence is shown in Figure 1 As shown, the nucleotide sequences of the upstream primer and the downstream primer for amplifying the molecular marker S617 are shown as SEQ ID No.2 and SEQ ID No.3, and the extension primer is shown as SEQ ID No.4 (Table 3).

[0043] Table 2 Molecular markers of CLASRP gene S617 related to glycogen content

[0044] Table 3 Amplification primers and extension primers for molecular markers .

[0045] Example 2 The step of using the molecular marker S617 provided by the present invention to breed the Omi oyster variety with high glycogen content trait is: extracting the Omi oyster sample DNA as a template, using the amplification primers S617 upstream primer (SEQ ID No. 2) and S617 downstream primer (SEQ ID No. 3) of the molecular marker S617 for PCR amplification, and the PCR amplification system is as follows: 0.1 μL dNTP, 0.4 μL MgCl2, 0.1 μL HotStar Taq (5 U / μl), 0.5 μL PCR buffer, 0.25 μL of each upstream and downstream primer (10 μmol / L), 1 μL DNA, 2.4 μL water. The PCR amplification conditions are: 94 ° C pre-denaturation for 3 min, 94 ° C denaturation for 30 s, 56 ° C annealing for 25 s, 72 ° C extension for 30 s, and a total of 40 cycles.

[0046] The obtained amplicon was treated with alkaline phosphatase to remove dNTPs, and then further extended. The extension reaction was performed as follows: 94 °C pre-denaturation for 30 s, 94 °C denaturation for 5 s, annealing and extension for 5 cycles, including 52 °C annealing for 5 s, and 80 °C extension for 5 s, for a total of 40 cycles. Reaction system: 2 μL EXTEND Mix, 7 μL SAP+PCR reactant. The extension reaction product was desalted and used for mass spectrometry detection. If the genotype of the 466th base of the molecular marker S617 in the test result was CC, the Omi oyster sample to be tested was an Omi oyster variety with high glycogen content.

[0047] The above implementation cases are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to replace some of the technical features therein by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions claimed to be protected by the present invention.

Claims

1. A molecular marker S617 for identifying the gene CLASRP associated with the glycogen content trait of Omi oysters, characterized in that: The nucleotide sequence of the molecular marker S617 is shown in SEQ ID No.

1.

2. The molecular marker S617 for identifying the gene CLASRP associated with the glycogen content trait of Omi oyster according to claim 1, characterized in that: The molecular marker S617 is located on chromosome 6, and the 466th base in the nucleotide sequence of the molecular marker S617 is T or C.

3. The molecular marker S617 for identifying the gene CLASRP associated with the glycogen content trait of Omi oyster according to claim 2, characterized in that: The genotype CC of the 466th base of the molecular marker S617 is a high glycogen content trait genotype.

4. The amplification primer and extension primer of the molecular marker S617 according to claim 1, characterized in that: The nucleotide sequences of the amplification primers are shown in SEQ ID No.2 and SEQ ID No.3, and the extension primer sequence is shown in SEQ ID No.

4.

5. Use of the molecular marker S617 according to claim 1 or the amplification primer and extension primer according to claim 4 in breeding a variety of Omi oysters with a high glycogen content trait.

6. The use according to claim 5, characterized in that: The steps of breeding the Omi oyster variety with the trait of high glycogen content are as follows: extracting DNA of a sample of Omi oyster to be tested as a template, performing PCR amplification using the amplification primers S617 upstream primer and S617 downstream primer of the molecular marker S617, treating the obtained amplicon with alkaline phosphatase to remove dNTPs, further performing extension reaction and desalting, and using the desalted product for mass spectrometry detection. If the genotype of the 466th base of the molecular marker S617 in the detection result is CC, then the Omi oyster sample to be tested is an Omi oyster variety with high glycogen content.

7. The use according to claim 6, characterized in that: The PCR amplification system was as follows: 0.1 μL dNTP, 0.4 μL MgCl2, 0.1 μL HotStar Taq 5 U / μl, 0.5 μL PCR buffer, 0.25 μL each of the upstream and downstream primers at 10 μmol / L, 1 μL DNA, and 2.4 μL water.

8. The use according to claim 6, characterized in that: The conditions for the PCR amplification were: 94°C pre-denaturation for 3 min, 94°C denaturation for 30 s, 56°C annealing for 25 s, 72°C extension for 30 s, for a total of 40 cycles; The conditions of the extension reaction are: pre-denaturation at 94°C for 30 s, denaturation at 94°C for 5 s, annealing and extension for 5 cycles including annealing at 52°C for 5 s and extension at 80°C for 5 s, for a total of 40 cycles.

9. A kit for breeding high glycogen content varieties of Omi oysters, characterized in that: The kit contains the molecular marker S617 according to claim 1 and / or the amplification primer and extension primer according to claim 4.

10. Use of the molecular marker S617 according to claim 1 in genetic diversity analysis, germplasm identification and genetic map construction of Crassostrea gigas.

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