CN104152444B - A kind of SNP marker related to long oyster glycogen content character and its application - Google Patents

Abstract

The present invention relates to a kind of SNP marker related to long oyster glycogen content character and its application.SNP marker site is named as TY202, and on the 3rd extron of glycogen debranching enzyme gene, genotype is Y.The TY202 is located at 5 ' the 7022nd bases of end of glycogen debranching enzyme gene.3rd extron is located at 5 ' 6,446 7043 base-pairs of end of glycogen debranching enzyme gene.SNP marker loci gene type is T/T or T/C individual glycogen content is higher than the individual that genotype is C/C.The method of present invention detection idiotype can predict the height of individual glycogen content, selection target individual be can be used in breeding, for molecular marker assisted selection breeding.The use of concentrated acid concentrated base in glycogen detection process is avoided, makes operation safer and simpler.

Description

A SNP marker related to glycogen content traits of long oyster and its application

technical field

The invention belongs to the technical fields of molecular biology and genetic breeding, in particular to a SNP marker related to the trait of glycogen content in long oyster.

Background technique

Long oysters belong to the clambranchia (or bivalves) in the mollusk phylum. The meat is tender and nutritious. Glycogen is an important flavor nutrient in oysters, which can be directly absorbed by the body, thereby reducing the burden on the pancreas. Therefore, it is very effective in the prevention and treatment of diabetes.

Glycogen debranching enzyme is an enzyme in the process of glycogen degradation in oysters. Its main function is to hydrolyze α-1,6 glycosidic bonds, remove branches of glycogen, and make branched glycogen into linear glycogen. Deficiency of human glycogen debranching enzyme can cause glycogen storage syndrome-type 111 disease. At present, the full-length DNA of glycogen debranching enzyme is available. Mutation screening has been carried out at home and abroad and many mutation sites have been found, but no recognized mutation hotspots have been found.

SNP is single nucleotide polymorphism (single nucleotide polymorphism), which mainly refers to the polymorphism of DNA sequence caused by the variation of a single nucleotide at the genome level. SNPs are widely distributed, with high density and high genetic stability. Compared with other types of molecular markers, SNP markers can better reflect the sequence information in the biological genome, and can study the association between sequence polymorphisms in the genome and phenotypic traits in more detail, effectively guiding gene identification and positioning work.

Aquatic animal breeding research started late and mainly focused on the study of growth traits. Most of the methods used traditional selection, hybridization, long cycle and slow effect. There are not many studies on the quality traits of long oyster. In recent years, with a series of breakthroughs in genomics research, molecular breeding techniques such as molecular marker-assisted selection have become increasingly mature, greatly improving the level of genetic breeding. The use of molecular marker-assisted selection can reduce blindness, shorten the breeding period, and improve the efficiency of breeding. Association analysis is a popular method developed in recent years to identify molecular markers and genes associated with target traits. Association analysis, also known as linkage disequilibrium mapping (LD mapping) or association mapping (Association Mapping), is an analysis method based on linkage disequilibrium to identify the relationship between target traits and genetic markers or candidate genes in a certain population. Association analysis can detect all recombination and mutation events in a specific population with strong precision.

Contents of the invention

The purpose of the present invention is to provide a SNP marker related to the glycogen content of long oyster, so as to provide a reference for molecular marker-assisted selection of long oyster.

To achieve the above object, the technical solution of the present invention is a SNP marker related to the glycogen content of long oyster, which is located on the third exon of the glycogen debranching enzyme gene, and the genotype is Y. The SNP site TY202 was obtained, which was located on the third exon at the 5' end 6446-7043 base pairs of the glycogen debranching enzyme gene. The TY202 is located at the 7022nd base at the 5' end of the glycogen debranching enzyme gene.

The specific primer sequence is: F: 5'-TAGTAAAGACAGGCAGCA-3'; R: 5'-TCATTTGTAGACAGGGAG-3'; the length of the amplified fragment of the primer is 75bp, and the amplified nucleotide sequence is shown in SEQ ID NO.1.

The genotype is T/T, T/C or C/C, and the glycogen content of individuals with genotype T/T or T/C is higher than that of individuals with genotype C/C at this locus.

The genotype and phenotype data of each individual were detected in the wild population, and the genotype and phenotype data were predicted by association analysis, and the correlation between TY202 and the glycogen content of the long oyster reached a significant level (P<0.05), which was significantly related to that of the long oyster. Trait associations of oyster glycogen content for marker-assisted selection breeding of oyster oysters.

The detection method of the SNP marker related to the trait of glycogen content in long oyster, the screening steps are:

a) Collect multiple individuals from the wild oyster population in Jiaonan, Qingdao, dissect them, take samples by tissue (adductor muscle, gill tissue, etc.), freeze them with liquid nitrogen and store them at -80°C as experimental materials;

b) Determining the glycogen content in the adductor muscle of each individual to obtain phenotypic data;

c) Extract the total DNA of each individual gill with phenol-chloroform extraction;

d) Select genes related to glycogen metabolism of oyster oyster as candidate genes, design primers according to the SNP sites predicted by the transcriptome data of oyster oyster, and use HRM method to detect individual genotypes for each SNP after screening the primers;

e) Using GAPIT software to conduct association analysis, predict the SNP loci related to the glycogen content traits of long oyster.

Advantages of the present invention:

The method for detecting individual genotypes in the invention can predict the level of individual glycogen content, and can be used in breeding to select target individuals and molecular marker-assisted selection breeding. It avoids the use of concentrated acid and alkali in the glycogen detection process, making the operation safer and easier.

Description of drawings:

Figure 1 is the result of primer screening. Among them, lane 5 is Marker, and the Marker used is Marker1. Swimming lanes 10-13 are the screening results of primers for SNP site TY202.

Figure 2 is the HRM verification result of SNP site TY202.

Figure 3 is the result of SNP site TY202 population typing.

detailed description

The following examples will further illustrate the present invention, and the examples are only used to illustrate the present invention, and the present invention is not limited thereto. The experimental supplies used in the present invention are all purchased from the market.

Sequence Listing (1) Information of SEQ ID NO.1

Sequence feature length: 75bp

Type: nucleic acid

Chain type: double chain

Topology: Linear

Molecule type: DNA

Source: Long Oyster

Sequence description:

TAGTAAAGACAGGCAGCATGCTGAYGGTGAAGGGTACTTTTTGGTGGACCCCATCCTCCTCCCTGTCTACAAAATGA

Example 1

Screening of SNP sites related to glycogen content

a) Collection of samples: A total of 144 wild oysters were collected from wild oyster populations in Jiaonan, Qingdao. They were dissected, and samples were taken by tissue (adductor muscle, gill tissue, etc.), and frozen in liquid nitrogen at -80°C. Save for later.

b) Detection of glycogen content: the relative content of glycogen in the adductor muscle of each individual was detected with the muscle glycogen and liver glycogen determination kit of Nanjing Jiancheng Bioengineering Institute, and the detection method was carried out according to the operation steps in the manual.

1) Sampling: Take the frozen muscle sample, rinse it with normal saline, blot it dry with filter paper, and weigh it (sample weight ≤ 100 mg).

2) Hydrolysis: according to the weight of the sample (mg): the volume of lye in the kit (μl) = 1:3, add them together to the test tube, cook in a boiling water bath for 20 minutes, cool in running water, and obtain a glycogen hydrolyzate.

3) Prepare the glycogen detection solution: dilute the glycogen hydrolyzate with distilled water to obtain the glycogen detection solution. The amount of distilled water added is (μl): sample weight (mg)*16.

The operation table is shown in Table 1.

Table 1

blank tube standard tube Assay tube Distilled water (ml) 1.0 0.9

0.01mg/ml standard (ml) 1.0 Glycogen detection solution (ml) 0.1 Chromogenic solution (ml) 2 2 2

Both the standard solution and the chromogenic solution are provided in the kit.

After mixing, boil in boiling water for 5 minutes. After cooling, use a blank tube to set zero and measure the OD value of each tube at a wavelength of 620 nm and an optical path of 1 cm.

Calculated as follows:

c) DNA extraction: the total DNA of gills of each individual was extracted by phenol-chloroform extraction.

1) Take a 1.5 ml centrifuge tube, add 700 μl DNA extraction buffer (100 mM Tris-HCl, 5 mM EDTA), take 3-10 mg of oyster gill tissue, and cut it into pieces with scissors. Utensils used must be flame sterilized and flushed with ddH2O between _two individuals to prevent cross-contamination between individuals. Add 35 μl SDS and mix well. Add 2 μl proteinase K and mix well.

2) Incubate the centrifuge tubes in a metal bath at 65°C, add 2 μl proteinase K to each tube after 1.5 hours, shake the centrifuge tubes gently during the period and continue to incubate for more than half an hour after the tissue is completely digested, the total incubation time is at least 3 hours.

3) Cool the incubated sample to room temperature, add an equal volume of PCI, mix thoroughly, and centrifuge at 13,000 rpm for 10 min. Repeat the above steps once. PCI is a mixed solution of phenol: chloroform: isoamyl alcohol (25:24:1).

4) The supernatant from step 3) was extracted once with chloroform:isoamyl alcohol (24:1), and centrifuged at 13000 rpm for 5 min.

5) Transfer the supernatant from step 4) into an equal volume of isopropanol at -20°C, mix well by inverting back and forth, and place at -20°C for 20 minutes.

6) The mixture in step 5) was centrifuged at 13000 rpm for 5 min at 4°C. Carefully decant all liquid, taking care not to dislodge or pour off the white pellet at the bottom.

7) Wash twice with 75% ethanol at -20°C and pour out the liquid, place a centrifuge tube at the opening to dry the ethanol,

When the white precipitate turns colorless and transparent, add 50-100 μl (depending on the amount of DNA) ddH2O to dissolve the DNA. Store at -20°C for later use.

d) Primer design: According to the SNP sites predicted from the transcriptome data of oyster oyster, Primerpremier 5 software was used to design primers, and the size of the amplified product was controlled at 50-100bp. The final primer sequence in this experiment was: F: 5'-TAGTAAAGACAGGCAGCA- 3'; R: 5'-

TCATTTGTAGACAGGGAG-3'. The length of the fragment amplified by the primers is 75bp, and the amplified nucleotide sequence is shown in SEQ ID NO.1.

e) Primer screening: The DNA of 4 individuals was randomly selected from the experimental population as a template for PCR amplification. The PCR reaction used 10 μl of the system and was sealed with 15 μl of mineral oil. The reaction conditions are shown in Table 2.

Table 2

The amplified product was detected by 10% non-denaturing polyacrylamide gel, and electrophoresis was performed at a voltage of 200V for 20 min. Observation results under UV gel imager (Figure 1). The electrophoresis band is clear and single and consistent among the four templates, indicating that the primer has good specificity and high amplification efficiency, and can be used in the next experiment; f) HRM method to verify the SNP site:

1) Randomly pick the DNA of 8 individuals from the experimental population as a template, and use the screened primers to perform PCR amplification again. The amplification reaction is carried out in a skirted 96-well PCR reaction plate, and the PCR reaction uses a 10 μl system , sealed with 15 μl mineral oil, and the reaction conditions are shown in Table 2.

2) After the reaction, add 1 μl of internal standard and 1 μl of LC-green dye, centrifuge briefly, denature at 95°C for 10 minutes, and cool to room temperature.

The internal standard is a short nucleotide sequence with a stable annealing temperature, 50 bp in length, and is composed of equal volumes of high-temperature internal standard and low-temperature internal standard.

3) Take out the 96-well PCR reaction plate and put it into the LightScanner 96 machine for HRM detection, collect the fluorescent signals between 55°C and 95°C, and analyze the results according to the melting curve after the operation is completed (Figure 2). The points with neat melting curves are for verification The selected SNP loci.

g) Detection of individual genotype by HRM method: 144 individuals in the experimental population were amplified by PCR using the screened qualified primers, and the melting curve of each individual was obtained ( FIG. 3 ). The specific operation method is the same as above.

h) Association analysis: Import phenotype data and genotype data into GAPIT software, and run the software for association analysis. The results of genome-wide prediction were obtained, and the SNP site TY202 (P=0.008) related to the trait of glycogen content in long oyster was screened out according to the results of genome-wide prediction, the 25th nucleus from the 5' end in SEQ ID NO.1 glycosides.

The average glycogen content of 144 individuals is obtained by counting the glycogen content of each individual. The average glycogen content of individuals whose genotype is T/T is 5.48mg/g, and the glycogen content of individuals whose genotype is T/C The average content is 5.59mg/g. The average glycogen content of individuals with genotype C/C was 4.12 mg/g. Therefore, the glycogen content of individuals with genotype T/T or T/C at this locus is higher than that of individuals with genotype C/C.

Example 2

Application of a SNP marker associated with glycogen content in long oyster

a) Collection of samples: A total of 96 wild oysters were collected in Shentanggou, Qingdao, and dissected, and the gills and adductor muscles were taken, and stored at -80°C after quick freezing with liquid nitrogen.

b) DNA extraction: the extraction method is the same as c) in Example 1.

c) SNP site TY202 genotype detection:

1) Using the DNA of 96 wild long oysters in Shentanggou as a template, PCR amplification was carried out with specific primers of TY202, and the amplification reaction was carried out in a skirted 96-well PCR reaction plate. Seal with mineral oil, and the reaction conditions are shown in Table 2.

2) After the reaction, add 1 μl of internal standard (internal standard as above) and 1 μl of LC-green dye, denature at 95°C for 10 minutes after brief centrifugation, and cool to room temperature.

3) Take out the 96-well PCR reaction plate and put it into the LightScanner 96 machine for HRM detection, collect the fluorescent signal between 55°C and 95°C, analyze the results according to the melting curve after the run, and count the genotype of each individual.

After detection, only the genotype of the 40th group is C/C, and the others are all genotypes of T/T or T/C. According to the conclusion of Example 1, the glycogen The content is lower than that of other groups, and then the detection of glycogen content is carried out.

d) Detection of glycogen content: the detection method is the same as d) in Example 1.

The data in Table 3 shows that the average glycogen content of individuals whose genotype is T/T at the SNP site TY202 is 6.11 mg/g, the average glycogen content of individuals whose genotype is T/C is 6.76 mg/g, and the average glycogen content of individuals whose genotype is C The individual glycogen content of /C was 2.50 mg/g. The above results proved that the average content of glycogen in individuals with genotype T/T or T/C was higher than that in individuals with genotype C/C.

After testing, our prediction result is correct, the glycogen content of individuals in the 40th group is lower than that of other groups.

To sum up, the SNP site TY202 is associated with the glycogen content. By detecting the genotype of the SNP site TY202, the glycogen content of an individual can be predicted.

table 3

individual number Glycogen content genotype individual number Glycogen content genotype individual number Glycogen content genotype 1 8.43 TT 33 2.19 TC 65 5.89 TT 2 5.98 TT 34 3.93 TT 66 8.35 TC 3 7.84 TC 35 6.36 TT 67 5.53 TT 4 4.50 TT 36 5.23 TT 68 6.42 TT 5 6.58 TT 37 4.58 TT 69 8.99 TC 6 2.85 TT 38 5.55 TT 70 9.71 TT 7 7.13 TT 39 5.46 TC 71 8.05 TC 8 8.44 TT 40 2.50 CC 72 6.75 TT 9 9.17 TT 41 4.31 TT 73 5.34 TC 10 6.53 TT 42 6.40 TT 74 4.50 TT 11 3.45 TT 43 8.04 TT 75 5.66 TT 12 6.08 TT 44 4.95 TT 76 4.43 TT 13 6.29 TT 45 5.53 TT 77 9.15 TT 14 6.85 TT 46 7.80 TT 78 6.79 TT 15 4.25 TT 47 7.63 TC 79 8.06 TT 16 8.19 TT 48 6.38 TT 80 5.08 TT 17 3.44 TT 49 6.19 TT 81 5.70 TT 18 3.47 TT 50 5.79 TT 82 7.62 TT 19 5.58 TT 51 6.90 TT 83 6.59 TT 20 5.99 TT 52 8.10 TT 84 5.89 TT twenty one 7.07 TC 53 7.55 TT 85 3.42 TC twenty two 7.84 TT 54 3.48 TT 86 10.20 TT twenty three 5.20 TT 55 4.50 TT 87 6.01 TT twenty four 4.00 TC 56 3.95 TT 88 9.12 TT 25 6.15 TT 57 4.23 TT 89 7.80 TT 26 4.05 TT 58 5.17 TT 90 8.66 TC 27 5.17 TC 59 5.91 TT 91 4.85 TT 28 5.25 TT 60 3.90 TT 92 5.31 TT 29 7.15 TT 61 6.91 TT 93 5.75 TT 30 2.19 TT 62 5.76 TT 94 5.65 TT 31 7.37 TT 63 10.11 TC 95 4.78 TT 32 5.37 TT 64 9.79 TT 96 6.20 TT

Note: The unit of glycogen content is mg/g.

Claims (1)

1. The application of a pair of SNP marker-specific primers related to the glycogen content of long oyster in the detection of glycogen content. The sequence of the specific primer pair is: F: 5'-TAGTAAAGACAGGCAGCA-3'; R: 5' -TCATTTGTAGACAGGGAG-3'; The SNP marker is located on the third exon of the glycogen debranching enzyme gene, and the genotype is Y.