A salinity regulation SNP marker, primer set, kit, identification method and application thereof for Omi oyster

Abstract

The present invention belongs to the field of genetic engineering and genetic breeding, and relates to a salinity-regulated SNP marker, primer set, kit, identification method and application thereof for Omi oysters. The salinity-regulated SNP marker for Omi oysters is located at base 13289957 of chromosome 5. The present invention provides a SNP marker significantly correlated with CDO1 gene expression, which has the advantage that the genotype of parental shellfish can be identified before seed breeding, thereby improving the tolerance of offspring to changes in salinity environment. The SNP marker obtained by the present invention has high credibility and stable results.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering and genetic breeding, and relates to a salinity regulation SNP marker, a primer set, a kit, an identification method and applications thereof for Crassostrea gigas. Background Art

[0002] Salinity is one of the important basic environmental factors that affect the life activities of aquatic organisms. Slight fluctuations in environmental salinity will affect the survival ability of organisms. Taurine, as a free amino acid, accounts for 47%-80% of the total free amino acids in oysters and has an important osmotic regulation effect. Through systematic evolutionary analysis, it was found that the key gene of the taurine synthesis pathway in oysters, the CDO1 gene, has undergone a significant expansion, and the expansion is particularly significant in the Omi oyster. At present, there is a lack of screening for the regulatory sites of the salinity adaptation gene CDO1. Summary of the invention

[0003] The invention aims at a salinity regulating SNP marker of Crassostrea gigas and application thereof in identifying salt-tolerant oyster individuals.

[0004] To achieve the above object, the technical solution adopted by the present invention is:

[0005] A salinity-regulating SNP marker for Crassostrea gigas. The SNP marker for expression regulation of the salinity adaptation gene CDO1 of Crassostrea gigas is located at base 13,289,957 on chromosome 5.

[0006] The SNP marker is the sequence shown in SEQ ID NO: 2; the 969th base in the sequence has a mutation type of A / G (cis regulatory site LG05: 13289957).

[0007] The genotype is homozygous or heterozygous, that is, the genotype is TT or CT.

[0008] An application of the Omi oyster salinity regulation SNP marker, and an application of the SNP marker in identifying salinity-tolerant oyster individuals.

[0009] A primer set for detecting the SNP marker, the primer set comprising a specific peripheral amplification primer and a single base extension primer gene sequence;

[0010] Specific peripheral amplification primers are:

[0011] LG05:13289957-F:5'-GACAAACAAAGCACCGAT-3';

[0012] LG05:13289957-R:5'-AAGACAGGCCGAGGG-3';

[0013] The single base extension primer gene sequence is:

[0014] 5'-TTTTTTTTTTTTTTTTTTTTTTTTTTTATCAGATAACTTTCATTAAATAGGT

[0015] ATCA-3'.

[0016] An application of the primer set, which is the application of the primer set in identifying salinity-tolerant oyster individuals.

[0017] A kit for detecting the SNP marker comprises the primer set.

[0018] A method for identifying salinity-tolerant oyster individuals comprises extracting genomic DNA of the oyster to be detected; using the extracted genomic DNA as a template, amplifying a sequence fragment containing a target SNP site using the primer set or the specific peripheral amplification primer in the kit, and then performing typing detection on the SNP site using the single base extension primer in the primer pair, thereby identifying the genotype of the oyster to be detected, and determining the dominant individual of the salinity-tolerant oyster according to the genotype; wherein the individual with the genotype of CT is the salinity-tolerant oyster individual.

[0019] Determine the SNP site of the oyster to be tested and perform typing detection:

[0020] a. The extracted DNA sample is diluted and used as a PCR template, and peripheral amplification is performed using specific primers;

[0021] PCR reaction system: DNA polymerase 22 μl, forward primer 1 μl, reverse primer 1 μl, template DNA 1 μl; the reaction program of PCR amplification is shown in Table 1:

[0022] Table 1 PCR amplification reaction procedure

[0023]

[0024] b. SNaPshot PCR: The PCR product was used as the template for SNaPshot PCR. After the product was purified, the site was detected using a single base extension primer;

[0025] Reaction system: 2 μl DNA polymerase, 2 μl primers, 1 μl purified PCR product; the reaction procedure is shown in Table 2:

[0026] Table 2 Reaction procedure

[0027]

[0028] c. Use the above amplification products to identify the genotype of each individual.

[0029] Beneficial effects of the present invention:

[0030] The present invention predicts the expression level of the salinity adaptation gene of individual oysters by detecting the individual genotype, can quickly screen oysters with different expression levels in large quantities, identify salt-tolerant oysters, and can detect the parental genotypes before breeding through non-destructive sampling technology and apply it to breeding, select individuals with target genotype combinations as parents, carry out molecular marker-assisted selection breeding, and breed progeny with high salt tolerance. It can also serve as a potential marker for predicting the adaptability potential of oysters under the current global warming environment.

[0031] The present invention conducts genome-wide association analysis (eGWAS) of gene expression on the key gene CDO1 for oyster salinity adaptation, and associates it with a trans regulatory site of a key salinity adaptation gene CDO1: LG05:13289957, which is located in the intergenic region of SMAD4. It may further regulate the expression level of the CDO1 gene by affecting the function, structure and expression of the SMAD4 gene; and obtains its optimal genotype, and uses its genotype to predict individuals with different expression levels of the salinity adaptation gene. The candidate markers of the present invention are obtained by liquid chip genotyping, obtained by eGWAS analysis, and further verified by other populations for the regulatory site. The results are reliable and can be used to screen individuals with different expression levels of the CDO1 gene, identify oyster parents with high salt tolerance, breed oyster individuals with high salt tolerance, and predict the potential for oyster environmental adaptation;

[0032] The method of the present invention can also be used to quickly screen oyster individuals with different expression levels in large quantities, and can identify the genotype of the parent shellfish before breeding, screen oyster parents with high salt tolerance, improve the salinity tolerance of the offspring, and predict the adaptability potential of oysters under current climate change. The results of the present invention are reliable, the SNP sites obtained are highly credible, and the effect is stable. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 It is a frequency distribution histogram of the CDO1 gene expression level of 278 oyster individuals in the TSF3 population provided by an embodiment of the present invention.

[0034] Figure 2 1 is an eGWAS analysis diagram of an embodiment of the present invention, a is a Manhattan diagram of the CDO1 gene eGWAS analysis provided by an embodiment of the present invention, and b is an LD-block diagram of the SNP site identified by the eGWAS analysis provided by an embodiment of the present invention.

[0035] Figure 3 This is a correlation diagram between the CDO1 gene expression level and the trans regulatory site LG05:13289957 genotype provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0036] The present invention is further explained below with reference to the examples, but the examples do not limit the present invention in any form.

[0037] The present invention provides SNP markers significantly associated with CDO1 gene expression, which has the advantage of being able to identify the genotype of parental shellfish before seed breeding, thereby improving the offspring's tolerance to environmental salinity changes. The SNP markers obtained in this study are highly credible and the results are stable. Specifically:

[0038] The southern Omi oyster population (TSF3) that has been domesticated in the north for two generations measured the expression level of the salinity adaptation candidate gene CDO1 in 278 individuals of Omi oysters, and then conducted genome-wide association studies (eGWAS). For the key regulatory modules and SNP sites located, association analysis was performed in another independent population (a hybrid family of northern and southern Omi oysters) to verify the results. Among them, the expression level of the CDO1 gene of each individual was detected in 278 oyster samples of the Omi oyster population. Through the eGWAS analysis of genotype and gene expression, it was associated with 2 cis-regulatory sites and 5 trans-regulatory sites (cis-eSNPs), forming two strongly linked regulatory modules. For one of the eSNPs, an association analysis was performed on 30 oyster individuals from another hybrid family of southern and northern Omi oysters (NSS): the LG05:13289957 site was found to be significantly correlated with the expression of the CDO1 gene (shown in the base sequence in SEQ ID NO:1) (p<0.05), and can be used to screen oyster individuals with strong salinity tolerance.

[0039] Example 1

[0040] The steps for screening SNP markers associated with salinity adaptation gene expression are:

[0041] A) Construction of experimental materials: In the spring of 2018, 30 wild male oysters and 30 wild female oysters were collected from Taishan, Guangzhou (N21°95′, E112°83′) to breed the first generation of southern population (TSF1). One month later, they were transferred to Qinzhou, Guangxi (N21°74′, E108°58′) (south) and Qingdao, Shandong (N36°57′, E120°95′) (north) for culture for about one year. In the spring of the second year, TSF1 oysters cultured in the northern sea area were used to breed TSF2 population. Similarly, TSF2 populations cultured in the northern sea area were used to breed TSF3 populations, and some of them were transferred to the north (Qingdao, QD) and the south (Qinzhou, QZ) for culture at the same time. In situ sampling of two generations of domesticated oysters (TSF3) was conducted, with a total of 278 northern-acclimated Crassostrea gigas cultured in a southern environment (QZ) and 91 cultured in a northern environment (QD) collected in December 2020. The third-generation population TSF3 was used for the screening of SNP markers associated with the expression of salinity adaptation genes.

[0042] B) Genotyping: Collect 278 offspring individuals from the above TSF3 population for targeted genotyping. For detailed steps, please refer to the article by Zhang et al. (Zhang et al. 2023):

[0043] 1. Genomic DNA shearing 2. Whole genome library construction 3. Probe hybridization capture 4. SNP detection.

[0044] C) Determination of gene expression: The expression of CDO1 gene was determined by qRT-PCR. The coding region of CDO1 gene is shown in SEQ ID NO:1. The frequency distribution histogram of CDO1 gene expression in 256 oysters in TSF3 population is shown in Figure 1 .

[0045] D) eGWAS analysis: The GAPIT software was used to perform genome-wide association (eGWAS) analysis using a compressed mixed effects model (GMLM) to determine the association between genotype and gene expression. In order to minimize false positives, the first ten PCA values ​​obtained from the population structure analysis were used as fixed effects, and the kinship matrix was used as a random effect in the mixed model to correct population stratification for more accurate results. A trans regulatory module and a cis regulatory module of the CDO1 gene were obtained, containing a total of 2 cis regulatory sites and 5 trans regulatory sites. The above 7 sites were verified in another independent population NSS, and only one candidate trans regulatory site LG05:13289957 was identified, which had different expression levels in different oysters. The site information can be found in the sequence SEQ ID NO:2. The Manhattan plot and LD-block plot of eGWAS are shown in Figure 2 .

[0046] From the above, the expression regulatory variation sites of the oyster salinity adaptation gene: CDO1 can be obtained. An expression regulatory module of the CDO1 gene was identified through eGWAS. For one of the eSNPs (LG05:13289957), the correlation between this site and the expression of the CDO1 gene was further verified by association analysis in another independent population NSS. A method for rapid genotype detection has been developed for this SNP site. The genotype (CT) of the LG05:13289957 site has a higher expression level of the CDO1 gene than the genotype (TT); subsequently, the genotype of the oyster individual can be identified to screen individuals with different expression levels of the salinity adaptation CDO1 gene, and then oyster parents with high salt tolerance can be screened for oyster breeding. In addition, it also provides basic research for predicting the adaptation potential of marine organisms under today's severe global climate change.

[0047] SEQ ID NO: 1 is the complete CDS sequence encoding the CDO1 gene of the Omi oyster, with a characteristic length of the information sequence of 948 bp and a gene ID of EVM0001666.1.

[0048] Information sequence features of SEQ ID NO: 1

[0049] Characteristic length: 948bp

[0050] Type: Nucleic Acid

[0051] Chain type: Single chain

[0052] Topology:

[0053] Molecule type: DNA

[0054] Source: Omi Oyster

[0055] Sequence Description:

[0056] 。

[0057] SEQ ID NO: 2 is the sequence of the upstream regulatory region of the gene that regulates the expression of the CDO1 gene, with a characteristic length of the information sequence: 2535 bp, LG05, 13288989 13291523.

[0058] Information sequence features of SEQ ID NO:2

[0059] Type: Nucleic Acid

[0060] Chain type: Single chain

[0061] Topology:

[0062] Molecule type: DNA

[0063] Source: Omi Oyster

[0064] Sequence Description:

[0065] GTATTTTTTTTTTTTATTTTTTTTTCATGGTGTAAGAAAGAAAAAGGTACAATCTTAGTCATCTTTTGCCCTGTTTTAAGAGAACTGGATGGTTGTAACCATTTTTAGACTTTATTTATCTCCATAAGAAGGAGAGCTTTATATTACTTGTATTAAGATGATATATAGAAAAACATTCAATTTTTTTTTTTTTAAAAGCTGAAATATGTAATAGAAAATTTTCTTTATTATATTTAAATAATAGTTTAAGGCTAAGAAATGAGATGTACAATTTCAAGGAAGATCTGATGGGCAATTTGTTGACCAACTAGCCACCTTAAAGGGGCATGGTCACAATTTTTTCTTCCAAAATTGCTGTTTAAATTTTAATATGTACAATAGGTCAAAATCAAAAATATTGTTTTTTTTTTTAACAAAAGTAACAATATTTTTTATTTGACCTATAGTGTACATGCTATAGTCTGGTCATTTGGCAATGTTAAAA ATATTTCCACTTAAAACTGATGACATCACAAATTTTTGCTAATTTTACTTAAAGTGTGGTGAAAAATAATATAATGAAAGAAATTGTCAATTGCCATGTTTACAAAAACACATTTGTTACTTAATGACGTGAGTTACATGCTTTAAACCTATACATGTACATGTTAAACATGTTAAAATAGATTGATAAACACACTTATTTTTTCATCTTGTTCCATGCATATGGACCATTTCTTCTAAAATGGGTCAAATAATTGTTGATTGTGGTGCTTTCTAACATTTGCTAAGATAAATCATTTATAAACTTTTCCCTAAAAAAAAAAAAAGTGGTTCCATTACTAAAGACAGAGAACTGATGAACATATTTGACAAACAAAGCACCGATAATATTTTGAGGTGTTAATTGCACATGTATGAACATGTTGAAATACATTGCATAAATAGATCATTTTAGTTATCAGATAACTTTCATTAAATAGGTATCA C

[0066] Example 2

[0067] The above-obtained sites were used to verify their association with CDO1 gene expression.

[0068] A) Pretreatment of experimental samples: The samples used were 4 mixed families NSS generated by hybridization of 4 southern and 4 northern Omiya oysters. The male parent was a 1-year-old wild Omiya oyster collected from Binzhou, Shandong (N38°18′, E118°05′), and the female parent was collected from Taishan, Guangzhou (N21°95′, E122°83′). After being temporarily raised in the farm for one month, the 4 families were mixed and cultured in a marine aquaculture base in Qinzhou. When the offspring oysters were 8 months old, in situ sampling began, that is, the offspring oysters were quickly placed in a large barrel filled with seawater and transferred to the shore farm for rapid sampling. The gill tissues of 30 mixed offspring individuals were collected, frozen in liquid nitrogen for temporary storage, and finally stored in a -80℃ ultra-low temperature refrigerator.

[0069] B) DNA extraction: DNA from 30 oysters was extracted according to the instructions of the Tiangen Marine Animal Tissue Genomic DNA Extraction Kit. DNA concentration was determined using a Nanodrop 2000 spectrophotometer. Quality was assessed using 1.0% gel electrophoresis.

[0070] C) Genotyping: SNaPshot (multiple single base extension technology) was used to detect the genotype of each individual target site (LG05:13289957).

[0071] Amplification primers were designed according to the upstream and downstream sequences of LG05:13289957 (sequence SEQ ID NO:2). The target fragment was amplified using the peripheral primers using the above-extracted DNA template. The obtained PCR product was then subjected to SNaPshot PCR using specific single-base primers to detect the genotype of the target site.

[0072] 1) Use specific peripheral primers to amplify the periphery of the SNP site:

[0073] Template: The extracted DNA sample was diluted to 20 ng / μl and used as a PCR template.

[0074] Primer sequences (peripheral primers):

[0075] LG05: 13289957-F: 5'-GACAACAAAGCACCGAT-3' (SEQ ID NO: 3);

[0076] LG05:13289957-R:5'-AAGACAGGCCGAGGG-3' (SEQ ID NO:4).

[0077] The reaction system is shown in Table 3:

[0078] Table 3 Reaction system

[0079]

[0080] The reaction procedure is shown in Table 4:

[0081] Table 4 Reaction procedure

[0082]

[0083] 2) Use specific primers to detect the SNP site:

[0084] SNaPshot PCR: In this reaction system, primer extension stops after one base.

[0085] Template: The above PCR products were purified and used as reaction templates for SNaPshot PCR.

[0086] Single base extension primer sequence:

[0087] 5'-TTTTTTTTTTTTTTTTTTTTTTTTTTTATCAGATAACTTTCATTAAATAGGT

[0088] ATCA-3' (SEQ ID NO:5).

[0089] The single base extension primer was diluted to 10 μM and SNaPshot PCR was performed.

[0090] The PCR system and components are shown in Table 5:

[0091] Table 5 PCR system and components

[0092]

[0093] The reaction procedure is shown in Table 6:

[0094] Table 6 Reaction procedure

[0095]

[0096] The amplified products were then subjected to capillary electrophoresis on a 3730 sequencer to identify the genotype of each individual at this marker (LG05:13289957). The specific capillary electrophoresis detection system is shown in Table 7:

[0097] Table 7 Capillary electrophoresis detection system

[0098]

[0099] After adding each component in sequence according to the above system, pre-denaturation was performed at 95°C for 5 minutes, and capillary electrophoresis was performed on a 3730 sequencer after cooling at -20°C. The results were analyzed using Gene mapper 4.1 software for accurate site data analysis, and genotyping was performed according to the corresponding peaks of the SNP sites.

[0100] D) RNA extraction: RNA was extracted using Tiangen RNAprep pure Tissue Kit. DNA concentration was determined using Nanodrop2000 spectrophotometer. Quality was assessed using 1.0% gel electrophoresis. Reverse transcription was used to obtain cDNA for subsequent quantitative experiments.

[0101] E) Quantitative PCR: Quantitative PCR primers were designed based on the sequence of CDO1 gene (SEQ ID NO: 1), and the experiment was carried out using the SYBR fluorescent quantitative kit of Nanjing Novozyme. The primer sequences are:

[0102] CDO1-F: 5'- GGTTGTGAAAGGCTATCGGATG-3' (SEQ ID NO: 6);

[0103] CDO1-R: 5'-TGTTCTATAAGATGTTCCTGTCGGC-3' (SEQ ID NO:7).

[0104] The reaction system is shown in Table 8:

[0105] Table 8 Reaction system

[0106]

[0107] The reaction procedure is shown in Table 9:

[0108] Table 9 Reaction procedure

[0109]

[0110] Omi elongation factor (CarEF) was used as an internal reference gene, and the gene expression level was normalized by the internal reference, and then 2 -△△ CT Methods The relative expression level of the target gene was analyzed and the data were logarithmically transformed. The CDO1 gene expression level of each progeny individual of the hybrid population of northern and southern Omi oysters NSS was obtained. The relevant information is shown in Table 10 below:

[0111] Table 10 COD1 gene expression

[0112]

[0113] E) Association analysis: The genotyping data of LG05:13289957 locus and CDO1 gene expression data of 30 oyster individuals were imported into Graph Primer software for association analysis, and the results of T-test were tested (see Table 11 and Figure 3 ):

[0114] Table 11 T-test results

[0115]

[0116] From Table 3 and Figure 3 It can be seen that the LG05:13289957 site is significantly correlated with the expression of the CDO1 gene (p<0.05), and the expression level of the CDO1 gene is: CT>TT; subsequently, the oyster individuals with high expression of the CDO1 gene can be selected by screening the individual genotype (CT).

[0117] As can be seen from the above, the genotype of oyster individuals can be identified through the SNP markers of the present invention to screen individuals that adapt to different expression levels of CDO1, and then screen oyster parents with high salinity tolerance for oyster breeding. In addition, it also provides basic research for predicting the adaptation potential of marine organisms under today's severe global climate change. The present invention provides a SNP marker that is significantly correlated with CDO1 gene expression, which has the advantage that the genotype of parent shellfish can be identified before seed breeding, thereby improving the offspring's tolerance to salinity environments. The SNP markers obtained in this study are highly credible and the results are stable.

Claims

1. A salinity regulation SNP marker for Omi oysters, characterized by: The SNP marker is the sequence shown in SEQ ID NO: 2; the 969th base in the sequence has a mutation type of A / G.

2. The salinity regulation SNP marker of Crassostrea gigas according to claim 1, characterized in that: The genotype is homozygous or heterozygous, that is, the genotype is TT or CT.

3. An application of the salinity regulation SNP marker of Crassostrea gigas according to claim 1, characterized in that: The SNP marker is used to identify salinity-tolerant Crassostrea gigas individuals, and individuals with a genotype of CT are salinity-tolerant Crassostrea gigas individuals.

4. A primer set for detecting the SNP marker according to claim 1, characterized in that: The primer set consisted of a specific peripheral amplification primer and a single-base extension primer gene sequence; Specific peripheral amplification primers are: LG05:13289957-F:5'-GACAAACAAAGCACCGAT-3'; LG05:13289957-R:5'-AAGACAGGCCGAGGG-3'; The single base extension primer gene sequence is: 5'-TTTTTTTTTTTTTTTTTTTTTTTTTTTATCAGATAACTTTCATTAAATAGGTATCA-3'.

5. An application of the primer set according to claim 4, characterized in that: The primer set is used to identify salinity-tolerant Crassostrea gigas individuals, and individuals with a genotype of CT are salinity-tolerant Crassostrea gigas individuals.

6. A kit for detecting the SNP marker according to claim 1, characterized in that: Comprising the primer set of claim 4.

7. A method for identifying individuals of salinity-tolerant Crassostrea gigas, characterized in that: Extract the genomic DNA of the tested Crassostrea gigas; use the extracted genomic DNA as a template, use the primer set described in claim 4 or the specific peripheral amplification primers in the kit described in claim 6 to amplify the sequence fragment containing the target SNP site, and then use the single base extension primer in the primer pair to perform typing detection on the SNP site, thereby identifying the genotype of the tested Crassostrea gigas, and determining the dominant individual of the salinity-tolerant Crassostrea gigas according to the genotype; wherein the individual with the genotype of CT is the salinity-tolerant Crassostrea gigas individual.

8. The method for identifying salinity-tolerant Crassostrea gigas individuals according to claim 7, characterized in that: Determine the SNP loci of the Omi oyster to be tested and perform typing detection: a. The extracted DNA sample is diluted and used as a PCR template, and peripheral amplification is performed using specific primers; b. SNaPshot PCR: The PCR product was used as the template for SNaPshot PCR. After the product was purified, the site was detected using a single base extension primer; c. Use the above amplification products to identify the genotype of each individual.

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