1. A Pm-SRB gene SNP molecular marker related to the carotene content of pinctada martensii shellfish, which is characterized by comprising:

a 1 st SNP molecular marker, wherein the 1 st SNP marker is a base A or a base G, the SNP marker is positioned at the 1102 th position of nucleic acid, and the nucleic acid has a nucleotide sequence shown as SEQ ID NO. 1;

a 2 nd SNP molecular marker, wherein the 2 nd SNP marker is a base A or a base G, the SNP marker is positioned at position 1143 of nucleic acid, and the nucleic acid has a nucleotide sequence shown in SEQ ID NO. 1;

a 3 rd SNP molecular marker, wherein the 3 rd SNP marker is a base T or C, the SNP marker is positioned at 1154 th position of nucleic acid, and the nucleic acid has a nucleotide sequence shown as SEQ ID NO. 1;

a 4 th SNP molecular marker, wherein the 4 th SNP marker is a base G or A, the SNP marker is positioned at 1642 th position of nucleic acid, and the nucleic acid has a nucleotide sequence shown in SEQ ID NO. 1.

2. The application of the SNP molecular marker of the SRB gene related to the carotene content of the pinctada martensii according to claim 1 in breeding parents of the pinctada martensii.

3. The application of the SNP molecular marker of the SRB gene related to the carotene content of the pinctada martensii according to claim 2, wherein the breeding parents of the pinctada martensii are parents with high carotenoid content and are screened for breeding filial generation groups.

4. The application of the SNP molecular marker of the SRB gene related to the carotene content of the pinctada martensii according to claim 2, which comprises the following steps:

(1) selecting individuals with mature gonads from the basic population as candidate parents, sampling adductor muscle, and extracting genome DNA;

(2) taking the extracted genome DNA as a template, performing PCR amplification by using a primer, performing sequence detection analysis, and determining the genotype of each SNP site of the candidate parent;

(3) analyzing haplotype gene combination of each candidate parent, and screening individuals of haplotype AATT as breeding parents.

5. The application of the SNP molecular marker of the SRB gene related to the carotene content of the pinctada martensii according to claim 4, wherein the primer sequence amplified by the PCR comprises SEQ ID 1.

6. The application of the SNP molecular marker of the SRB gene related to the carotene content of the pinctada martensii according to claim 4, wherein the method for extracting the genomic DNA in the step (1) comprises the following steps:

(1) taking 0.1g of adductor muscle tissue, shearing the tissue, adding artificially prepared seawater, mixing, centrifuging for 2min at 4000r/min, removing supernatant, adding 200 μ l buffer solution, and mixing;

(2) adding 400 mul of lysate and 10 mul of proteinase K into 200 mul of sample, mixing uniformly, placing in a 55 ℃ constant temperature water bath for 4-5h, adding 600ml of chloroform after digestion is completed, centrifuging for 5min at the rotating speed of 12000r/min, sucking 400 mul of supernatant into a sterilized 1.5ml centrifuge tube, adding 500 mul of precipitation solution, mixing uniformly, standing for 2min, centrifuging for 5min at the rotating speed of 12000r/min, discarding the supernatant, immediately adding 100 mul of NaCl solution with the concentration of 1.2mol/L, and shaking gently to dissolve DNA precipitation completely;

(3) adding RNase 6 μ l, water bathing at 37 deg.C for 5-10 min, pre-cooling at-20 deg.C, adding glacial ethanol 300 μ l, mixing, standing at-20 deg.C for 10min, centrifuging at 10000r/min for 5-8min, pouring off ethanol, washing with 70% glacial ethanol 300 μ l once, centrifuging at 10000r/min for 5min, pouring off ethanol, and drying on filter paper at room temperature for 30 min;

(4) the DNA was dissolved in 200. mu.l of double distilled water and stored in portions at 4 ℃.

7. The use of SNP molecular markers of SRB genes related to the carotene content of Pinctada martensii according to claim 4, wherein the analysis of haplotype gene combination in the step (3) requires analysis by correlating the carotenoid content.

8. The application of the SNP molecular marker of the SRB gene related to the carotene content of the pinctada martensii according to claim 7, wherein the carotenoid content needs to be tested by the following method:

(1) selecting individuals with mature gonads from the basic population as candidate parents, shearing adductor muscles, and drying in a vacuum freeze dryer for 36 hours until the weight of the tissues is not changed any more;

(2) grinding 0.1g of dried tissue into powder by using a mortar, placing the powder into a 15mL centrifuge tube, adding 5mL of analytically pure acetone, shaking the powder at the rotating speed of 200r/min for 2 hours in a dark place at room temperature, centrifuging the powder at room temperature for 10min, and taking supernatant;

(3) repeating the step (2) once, combining the two extracting solutions, measuring the light absorption value, and calculating the carotenoid content.

Background

Carotenoids are generally referred to as the two major pigments, the hydrocarbon of C40 and their oxidized derivatives. Carotenoids are bright in color and strong in coloring power, and have been widely used in colorants and feed additives. Besides being used as functional food, the food also has antioxidant function, such as oxygen radical removal, antioxidation, aging delay and the like. In recent years, studies on carotenoids have been gradually shifted to animals from the aspects of higher plants, algae and microorganisms, and some progress has been made. The fat-soluble organic compounds which play important physiological roles in animal bodies contribute to enhancing the immunity of the animal bodies, and play an important role in preventing cancers, cardiovascular diseases, osteoporosis and other chronic diseases.

With the development of science and technology, the research on carotenoid metabolic mechanism is continuously and deeply carried out. The research preliminarily shows that all tissues in the body of the marine shellfish contain rich carotenoid, the stress resistance of the shellfish has obvious correlation with the carotenoid content in the body, for example, the new variety of the Japanese scallop, namely the Japanese scallop, has better stress resistance because of being rich in the carotenoid compared with the common scallop. Earlier researches find that the carotenoid content in pinctada martensii is obviously different, and the individual survival rate of the pinctada martensii with high carotenoid content in the pearl harvesting period is higher. Therefore, how to effectively screen molecular markers obviously related to the carotenoid to obtain the genotype with high carotenoid content provides guidance for breeding new pinctada martensii strains.

Disclosure of Invention

The invention aims to provide a SNP (Single nucleotide polymorphism) marking method of a Pm-SRB gene related to the carotenoid content of pinctada martensii and application thereof.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

an SNP molecular marker of SRB gene related to the carotene content of pinctada martensii comprises:

a 1 st SNP molecular marker, wherein the 1 st SNP marker is a base A or a base G, the SNP marker is positioned at the 1102 th position of nucleic acid, and the nucleic acid has a nucleotide sequence shown as SEQ ID NO. 1;

a 2 nd SNP molecular marker, wherein the 2 nd SNP marker is a base A or a base G, the SNP marker is positioned at position 1143 of nucleic acid, and the nucleic acid has a nucleotide sequence shown in SEQ ID NO. 1;

a 3 rd SNP molecular marker, wherein the 3 rd SNP marker is a base T or C, the SNP marker is positioned at 1154 th position of nucleic acid, and the nucleic acid has a nucleotide sequence shown as SEQ ID NO. 1;

a 4 th SNP molecular marker, wherein the 4 th SNP marker is a base G or A, the SNP marker is positioned at 1642 th position of nucleic acid, and the nucleic acid has a nucleotide sequence shown in SEQ ID NO. 1.

A Pm-SRB gene SNP molecular marker related to the content of carotene in pinctada martensii shellfish is applied to the breeding parents of the pinctada martensii shellfish.

Furthermore, the Pinctada martensii breeding parent is a parent with high carotenoid content which is screened and used for breeding a filial generation population.

Further, the application of the Pm-SRB gene SNP molecular marker related to the carotene content of the pinctada martensii shellfish specifically comprises the following steps:

(1) selecting individuals with mature gonads from the basic population as candidate parents, sampling adductor muscle tissues, and extracting genome DNA;

(2) taking the extracted genome DNA as a template, performing PCR amplification by using a primer, performing sequence detection analysis, and determining the genotype of each SNP site of the candidate parent;

(3) analyzing haplotype gene combination of each candidate parent, and screening individuals of haplotype AATT as breeding parents.

Further, the primer sequence of PCR amplification.

Further, the method for extracting genomic DNA in the step (1) comprises the following steps:

(1) taking 0.1g of adductor muscle tissue, shearing the tissue, adding artificially prepared seawater, mixing, centrifuging for 2min at 4000r/min, removing supernatant, adding 200 μ l buffer solution, and mixing;

(2) adding 400 mul of lysate and 10 mul of proteinase K into 200 mul of sample, mixing uniformly, placing in a 55 ℃ constant temperature water bath for 4-5h, adding 600ml of chloroform after digestion is completed, centrifuging for 5min at the rotating speed of 12000r/min, sucking 400 mul of supernatant into a sterilized 1.5ml centrifuge tube, adding 500 mul of precipitation solution, mixing uniformly, standing for 2min, centrifuging for 5min at the rotating speed of 12000r/min, discarding the supernatant, immediately adding 100 mul of NaCl solution with the concentration of 1.2mol/L, and shaking gently to dissolve DNA precipitation completely;

(3) adding RNase 6 μ l, water bathing at 37 deg.C for 5-10 min, pre-cooling at-20 deg.C, adding glacial ethanol 300 μ l, mixing, standing at-20 deg.C for 10min, centrifuging at 10000r/min for 5-8min, pouring off ethanol, washing with 70% glacial ethanol 300 μ l once, centrifuging at 10000r/min for 5min, pouring off ethanol, and drying on filter paper at room temperature for 30 min;

(4) the DNA was dissolved in 200. mu.l of double distilled water and stored in portions at 4 ℃.

Further, the haplotype gene combination analysis process in step (3) requires analysis by correlating carotenoid content.

Further, the carotenoid content needs to be tested by the following method:

(1) selecting individuals with mature gonads from the basic population as candidate parents, shearing adductor muscles, and drying in a vacuum freeze dryer for 36 hours until the weight of the tissues is not changed any more;

(2) grinding 0.1g of dried tissue into powder by using a mortar, placing the powder into a 15mL centrifuge tube, adding 5mL of analytically pure acetone, shaking the powder at the rotating speed of 200r/min for 2 hours in a dark place at room temperature, centrifuging the powder at room temperature for 10min, and taking supernatant;

(3) repeating the step (2) once, combining the two extracting solutions, measuring the light absorption value, and calculating the carotenoid content.

Compared with the prior art, the invention has the beneficial effects that: according to the invention, SNP molecular markers are carried out on the mutation sites of the nucleotide sequence shown by SEQ ID NO. 1 of the pinctada martensii, and haplotype analysis is carried out on the SNP sites of the gene sequence which is obviously related to the carotenoid content, so that the carotenoid content of the haplotype AAAT is obviously higher than that of the GAAT and AGGC haplotypes. The method improves the accuracy of screening the Pinctada martensii parents, and can obtain the Pinctada martensii parents with high carotenoid content and stable heredity.

Detailed Description

Example 1

(one) sampling and processing

1. Genomic DNA extraction

Individuals were collected from the northern sea 28064j, the continental island geographical population as parents to breed 1 basal population. Randomly selecting 220 individuals from a breeding group with the age of 2, shearing adductor muscle, putting each in a 2mL centrifuge tube, adding 75% ethanol for fixation, putting in liquid nitrogen for quick freezing, and storing at-80 ℃.

Taking 0.1g of adductor muscle tissue, shearing the tissue, adding artificially prepared seawater, mixing, centrifuging for 2min at 4000r/min, removing supernatant, adding 200 μ l buffer solution, and mixing; adding 400 mul of lysate and 10 mul of proteinase K into 200 mul of sample, mixing uniformly, placing in a 55 ℃ constant temperature water bath for 4-5h, adding 600ml of chloroform after digestion is completed, centrifuging for 5min at the rotating speed of 12000r/min, sucking 400 mul of supernatant into a sterilized 1.5ml centrifuge tube, adding 500 mul of precipitation solution, mixing uniformly, standing for 2min, centrifuging for 5min at the rotating speed of 12000r/min, discarding the supernatant, immediately adding 100 mul of NaCl solution with the concentration of 1.2mol/L, and shaking gently to dissolve DNA precipitation completely; adding RNase 6 μ l, water bathing at 37 deg.C for 5-10 min, pre-cooling at-20 deg.C, adding glacial ethanol 300 μ l, mixing, standing at-20 deg.C for 10min, centrifuging at 10000r/min for 5-8min, pouring off ethanol, washing with 70% glacial ethanol 300 μ l once, centrifuging at 10000r/min for 5min, pouring off ethanol, and drying on filter paper at room temperature for 30 min; the DNA was dissolved in 200. mu.l of double distilled water and stored in portions at 4 ℃.

2. Total carotenoid content detection

(1) Drying the frozen and preserved adductor muscle in a vacuum freeze dryer for 36 hours until the weight of the tissue does not change any more;

(2) grinding 0.1g of dried tissue into powder by using a mortar, placing the powder into a 15mL centrifuge tube, adding 5mL of analytically pure acetone, shaking and extracting for two hours at 200r/min in the dark at room temperature, then centrifuging for 10min at room temperature, and taking supernatant;

(3) repeating the operation in the step (2) once, combining the two extracting solutions, and measuring the light absorption value.

The determination of the total carotenoid content is based on the following equation:

total carotenoid content (μ G/G) ═ a × K × V/E × G, where a is the absorbance value; k is a constant number (10)⁴) (ii) a V is the extract acetone volume (mL); e is the molar extinction coefficient (2500); g is the adductor muscle mass (G).

(II) detection of SNP molecular markers

1. Primer design

Primer sequences were designed using Primer 3online based on the pinctada martensii whole genome sequence, as in table 1.

TABLE 1 primer sequences

2. PCR amplification and detection

And amplifying the DNA of the sample by using the designed primer sequence, and detecting the genotype of the SNP locus of the amplified gene fragment. The method specifically comprises the following steps: denaturing the double-stranded DNA library into single strands using NaOH, synthesizing first strands; and (3) performing 35-cycle PCR amplification by using the synthesized first strand as a template, and sequencing and analyzing an amplification product.

The reaction system used for the SNP locus amplification is as follows: mu.L of 10 XBuffer, 0.8. mu.L dNTP (2.5mM), 2. mu.L DNA, 1. mu.L of 1. mu.M upstream and downstream primers, 0.1. mu.L enzyme (5U/. mu.L) and 3.1. mu.L sterile double distilled water.

The amplification conditions were: pre-denaturation at 95 ℃ for 15 min; denaturation at 94 ℃ for 30s, annealing at 90s, extension at 72 ℃ for 30s, 35 cycles; extension at 72 ℃ for 10 mins.

(III) SNP locus genotype and trait association analysis

A linear model is constructed by using a GLM method to perform correlation analysis of SNP locus typing data and carotenoid content, wherein the specific model is Y ═ mu + Gi + eij, Y is a character observed value, mu is an average number, Gi is an SNP effect value, and eij is a random error.

Haploview was used to perform haplotype analysis on significantly related SNP sites.

The results showed that a total of 4 SNPs in the exon region were significantly associated with carotenoid status (P < 0.05). The results of the correlation analysis of the gene frequency of the SNP site and the carotenoid content are shown in Table 2.

TABLE 2 Pm-SRB exon region SNP locus and carotenoid content correlation analysis

Note: different superscript letters at the same SNP site indicate significant differences between the different genotypes (P < 0.05). "-" indicates that the genotype individuals were less than 5% of the total number of individuals and were not involved in multiple comparisons.

As can be seen from Table 2, the carotenoid content of the individual with the SNP locus being AATT genotype is obviously greater than that of other genotypes, and it can be seen that the locus AATT of the nucleotide sequence of the gene is obviously related to the carotenoid content, so that the breeding new strain rich in the carotenoid content is cultivated by selecting the individual with the genotype combination AATT/AATT as a breeding parent.

The above embodiments are preferred embodiments of the present invention, and all similar processes and equivalent variations to those of the present invention should fall within the scope of the present invention.

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