Method for screening molecular marker of dna methylation of large yellow croaker and qrt-pcr primer

Abstract

The application discloses a screening method of DNA methylation molecular markers of Larimichthys crocea and qRT-PCR primers, and belongs to the technical field of molecular markers. The method and the qRT-PCR primers for screening the DNA methylation molecular markers of the Larimichthys crocea can screen genes with significant methylation and qRT-PCR difference, and then can be applied to Larimichthys crocea breeding, breeding research assistance, and can effectively improve the breeding rate of excellent varieties of the Larimichthys crocea.

Description

[0001] The present application is a divisional application of the parent application with the application number 202110724168.7, the filing date of June 29, 2021, and the name of “Molecular marker of DNA methylation of Larimichthys crocea and its application in breeding”. TECHNICAL FIELD

[0002] The present application belongs to the technical field of molecular markers, and specifically relates to a method for screening a molecular marker of DNA methylation of Larimichthys crocea and a qRT-PCR primer. BACKGROUND

[0003] Larimichthys crocea is a unique marine economic fish in China, and its breeding and seed production ranks first in the country, playing an important role in the marine aquaculture industry in China. At present, the Larimichthys crocea industry has entered a stage of transformation and upgrading, and the industry is increasingly eager for excellent varieties (lines) with fast growth, strong stress resistance and good quality. Therefore, Larimichthys crocea breeding has become an important link in the development of the Larimichthys crocea industry. Molecular breeding technology is a modern biological technology breeding method newly developed to overcome the problems of low efficiency and long cycle of traditional breeding techniques, and has been widely used in fish breeding. For example, Dong Zaijie et al. successfully bred a new variety of Cyprinus carpio (Fufei carp) using PIT marking technology and BLUP method; Li Shengjie et al. bred a new variety of Micropterus salmoides (Lacépède) (Yulai No. 1) with significant growth advantage using molecular biology technology combined with traditional breeding techniques; Zou Guiwei et al. successfully bred a new variety of Changfeng silver carp with fast growth and good body shape by comprehensively using gynogenesis, molecular markers and population breeding techniques; the new variety of Carassius auratus gibelio (Xinke No. 5) bred by the Institute of Hydrobiology, Chinese Academy of Sciences is a combination of population breeding techniques, gynogenesis and SSR molecular marker assisted breeding techniques, and has the characteristics of significant growth advantage and strong resistance to viruses and myxosporean. These research results show that molecular marker assisted breeding as a modern biological technology breeding means has great development potential and application prospect.

[0004] Epigenetics is one of the research hotspots in the field of molecular biology in recent years. Unlike the classic genetics theory that the inheritance of phenotypic characteristics is mainly determined by DNA base sequence, epigenetic regulation refers to the phenomenon that the expression of genes is stably inherited without substantial changes in DNA sequence, thereby leading to changes in phenotypic characteristics and potential reversibility, reflecting the relationship between genotype and phenotype. Therefore, by studying the epigenetic regulation mechanism of Pseudosciaena crocea, a foundation can be laid for selecting excellent varieties (lines) of Pseudosciaena crocea. However, the research on the molecular regulation mechanism and genetic basis of growth traits of Pseudosciaena crocea is not sufficient, and the transcriptome and epigenetic regulation mechanism of the growth axis at the whole level have not been reported.

[0005] The epigenetic related regulation mechanism mainly includes the following three aspects: DNA methylation, non-coding RNA action and histone modification. Among them, DNA methylation refers to the process of covalently transferring the methyl group on the S-adenosyl methionine molecule to the 5th carbon atom of the cytosine CpG dinucleotide by the catalytic action of DNA methyltransferase, forming 5-methylcytosine (5-mC). DNA methylation is one of the important modification methods of epigenetic regulation. The methylation modification of specific gene regions of genomic DNA can have important influence on chromosome conformation, gene expression regulation, genome defense, genome imprinting, etc. The CpG methylation located in the promoter region and enhancer region is negatively correlated with the transcriptional activity of genes, while the high methylation in the main body region of the gene is positively correlated with the expression of the gene. At the same time, organisms can form different degrees of methylation types according to the changes of the environment, thereby producing different phenotypes to adapt to the new environment, and can be inherited to the next generation. DNA methylation is an important method for studying the differences in growth traits of species.

[0006] Therefore, according to the genetic information of Pseudosciaena crocea, the difference characteristics of the transcriptome, genome and epigenetics of the population with significant difference in growth traits of Pseudosciaena crocea are studied, and a DNA methylation molecular marker which can be applied to the breeding of Pseudosciaena crocea and then obtain excellent genetic traits of excellent varieties (lines) becomes a technical problem to be solved at present. SUMMARY

[0007] In order to overcome the defects of the prior art, the technical problem to be solved by the present application is to provide a method for screening DNA methylation molecular markers which can be applied to the breeding of excellent Pseudosciaena crocea varieties (lines) and qRT-PCR primers.

[0008] In order to solve the above technical problems, a technical scheme adopted by the present application is as follows: a method for screening DNA methylation molecular markers of Pseudosciaena crocea, comprising the following steps:

[0009] Step 1, screening the extremely large individual group and the extremely small individual group with significant difference in growth traits from the breeding population under the same breeding condition;

[0010] Step 2, performing whole genome bisulfite sequencing on the extremely large individual group and the extremely small individual group to obtain DNA methylation genetic map at the whole genome level, screening the differentially methylated regions of the two populations, and obtaining growth axis functional genes with differential methylation therefrom;

[0011] Step 3, designing qRT-PCR primers and using qRT-PCR technology to detect the correlation between mRNA expression of the growth axis functional genes with differential methylation and methylation, and screening the differentially methylated functional genes;

[0012] Step 4, using primers for detecting DNA methylation molecular markers of large yellow croaker to verify the correlation between single gene DNA methylation level and growth traits of the differentially methylated functional genes, obtain DNA methylation sites with significant regulatory effect on the expression level of growth functional genes, and obtain DNA methylation molecular markers of large yellow croaker;

[0013] The DNA methylation molecular markers of large yellow croaker include a molecular marker fgf11, a molecular marker fgfrs2, a molecular marker egflp7 and a molecular marker pdgfrα.

[0014] The nucleotide sequence of the molecular marker fgf11 is SEQ ID No. 1.

[0015] The nucleotide sequence of the molecular marker fgfrs2 is SEQ ID No. 2.

[0016] The nucleotide sequence of the molecular marker egflp7 is SEQ ID No. 3.

[0017] The nucleotide sequence of the molecular marker pdgfrα is SEQ ID No. 4.

[0018] The primers for detecting DNA methylation molecular markers of large yellow croaker include:

[0019] The fgf11 upstream primer with the nucleotide sequence of SEQ ID No. 5;

[0020] The fgf11 downstream primer with the nucleotide sequence of SEQ ID No. 6;

[0021] The fgfrs2 upstream primer with the nucleotide sequence of SEQ ID No. 7;

[0022] The fgfrs2 downstream primer with the nucleotide sequence of SEQ ID No. 8;

[0023] egflp7 upstream primer with the nucleotide sequence of SEQ ID No. 9;

[0024] egflp7 downstream primer with the nucleotide sequence of SEQ ID No. 10;

[0025] pdgfrα upstream primer with the nucleotide sequence of SEQ ID No. 11;

[0026] pdgfrα downstream primer with the nucleotide sequence of SEQ ID No. 12.

[0027] Another technical solution adopted by the present application is a qRT-PCR primer for screening the DNA methylation molecular marker of Pseudosciaena crocea, comprising:

[0028] fgf11 upstream primer with the nucleotide sequence of SEQ ID No. 13;

[0029] fgf11 downstream primer with the nucleotide sequence of SEQ ID No. 14;

[0030] fgfrs2 upstream primer with the nucleotide sequence of SEQ ID No. 15;

[0031] fgfrs2 downstream primer with the nucleotide sequence of SEQ ID No. 16;

[0032] egflp7 upstream primer with the nucleotide sequence of SEQ ID No. 17;

[0033] egflp7 downstream primer with the nucleotide sequence of SEQ ID No. 18;

[0034] pdgfrα upstream primer with the nucleotide sequence of SEQ ID No. 19;

[0035] pdgfrα downstream primer with the nucleotide sequence of SEQ ID No. 20;

[0036] The DNA methylation molecular marker of Pseudosciaena crocea comprises a molecular marker fgf11, a molecular marker fgfrs2, a molecular marker egflp7 and a molecular marker pdgfrα;

[0037] The nucleotide sequence of the molecular marker fgf11 is SEQ ID No. 1;

[0038] The nucleotide sequence of the molecular marker fgfrs2 is SEQ ID No. 2;

[0039] The nucleotide sequence of the molecular marker egflp7 is SEQ ID No. 3;

[0040] The nucleotide sequence of the molecular marker pdgfrα is SEQ ID No. 4.

[0041] The method for screening the DNA methylation molecular marker of the big yellow croaker provided by the present application is based on the genetic information of the big yellow croaker genome, and combines high-throughput RNA-Seq, genome resequencing and bisulfite sequencing technology to carry out the difference characteristic research on the epigenetics of the big yellow croaker growth trait significantly different population, screen the differential methylation characteristics of the genes at the whole genome level, and apply the screened molecular marker to the production practice to assist the big yellow croaker breeding.

[0042] The qRT-PCR primer provided by the present application is used for screening the DNA methylation molecular marker of the big yellow croaker, and by using the primer in qRT-PCR, the expression levels of the genes in different phenotype trait populations in the same family are detected by using the qRT-PCR technology, the correlation between the mRNA expression of the growth axis functional gene with differential methylation and methylation is detected, the genes with significant methylation and qRT-PCR difference are screened from the sequencing results, and the screened molecular marker is applied to the production practice to assist the big yellow croaker breeding, thereby providing an important reference for the molecular marker assisted breeding and epigenetic regulation mechanism of the big yellow croaker. BRIEF DESCRIPTION OF DRAWINGS

[0043] Figure 1 Fig. 1 shows the methylation level distribution statistical diagram of methylation C of the L population of Example 1 of the specific embodiment of the present application;

[0044] Figure 2 Fig. 2 shows the methylation level distribution statistical diagram of methylation C of the S population of Example 1 of the specific embodiment of the present application;

[0045] Figure 3 Fig. 3 shows the DNA methylation level diagram in different transcription elements of the L population of Example 1 of the specific embodiment of the present application;

[0046] Figure 4 Fig. 4 shows the DNA methylation level diagram in different transcription elements of the L population of Example 1 of the specific embodiment of the present application;

[0047] Figure 5 Fig. 5 shows the methylation level verification result of the fgf11 gene of Example 1 of the specific embodiment of the present application;

[0048] Figure 6 Fig. 6 shows the methylation level verification result of the fgfrs2 gene of Example 1 of the specific embodiment of the present application;

[0049] Figure 7Fig. 1 shows the methylation level verification results of the egflp7 gene of Example 1 of the specific embodiment of the present application;

[0050] Figure 8 Fig. 2 shows the methylation level verification results of the pdgfrα gene of Example 1 of the specific embodiment of the present application;

[0051] Figure 9 Fig. 3 shows the gene amplification results of the fgfrs2 gene of Example 1 of the specific embodiment of the present application;

[0052] Figure 10 Fig. 4 shows the gene amplification results of the fgf11 gene of Example 1 of the specific embodiment of the present application;

[0053] Figure 11 Fig. 5 shows the gene amplification results of the pdgfrα gene of Example 1 of the specific embodiment of the present application;

[0054] Figure 12 Fig. 6 shows the gene amplification results of the egflp7 gene of Example 1 of the specific embodiment of the present application. DETAILED DESCRIPTION

[0055] To make the technical content, the purposes and effects of the present application clear, the following will be described in combination with the embodiments and the accompanying drawings.

[0056] The DNA methylation molecular marker of Pseudosciaena crocea of the present application, as shown in Table 1, includes the molecular marker fgf11, the molecular marker fgfrs2, the molecular marker egflp7 and the molecular marker pdgfrα;

[0057] The nucleotide sequence of the molecular marker fgf11 is shown in SEQ ID No. 1;

[0058] The nucleotide sequence of the molecular marker fgfrs2 is shown in SEQ ID No. 2;

[0059] The nucleotide sequence of the molecular marker egflp7 is shown in SEQ ID No. 3;

[0060] The nucleotide sequence of the molecular marker pdgfrα is shown in SEQ ID No. 4.

[0061] Further, the primers for detecting the above-mentioned DNA methylation molecular marker of Pseudosciaena crocea, as shown in Table 1, include:

[0062] The fgf11 upstream primer F1 with the nucleotide sequence shown in SEQ ID No. 5;

[0063] fgf11 downstream primer R1 with nucleotide sequence as shown in SEQ ID No. 6;

[0064] fgfrs2 upstream primer F1 with nucleotide sequence as shown in SEQ ID No. 7;

[0065] fgfrs2 downstream primer R1 with nucleotide sequence as shown in SEQ ID No. 8;

[0066] egflp7 upstream primer F1 with nucleotide sequence as shown in SEQ ID No. 9;

[0067] egflp7 downstream primer R1 with nucleotide sequence as shown in SEQ ID No. 10;

[0068] pdgfrα upstream primer F1 with nucleotide sequence as shown in SEQ ID No. 11;

[0069] pdgfrα downstream primer R1 with nucleotide sequence as shown in SEQ ID No. 12.

[0070] Further, the primers for screening the above-mentioned DNA methylation molecular markers of Pseudosciaena crocea are shown in Table 1, which include:

[0071] fgf11 upstream primer F2 with nucleotide sequence as shown in SEQ ID No. 13;

[0072] fgf11 downstream primer R2 with nucleotide sequence as shown in SEQ ID No. 14;

[0073] fgfrs2 upstream primer F2 with nucleotide sequence as shown in SEQ ID No. 15;

[0074] fgfrs2 downstream primer R2 with nucleotide sequence as shown in SEQ ID No. 16;

[0075] egflp7 upstream primer F2 with nucleotide sequence as shown in SEQ ID No. 17;

[0076] egflp7 downstream primer R2 with nucleotide sequence as shown in SEQ ID No. 18;

[0077] pdgfrα upstream primer F2 with nucleotide sequence as shown in SEQ ID No. 19;

[0078] pdgfrα downstream primer R2 with nucleotide sequence as shown in SEQ ID No. 20.

[0079] Table 1

[0080]

[0081]

[0082]

[0083]

[0084] From the above description, the beneficial effects of the present application are that the DNA methylation molecular marker of the present application can be applied to production practice for assisted breeding research, can be used for screening of parent fish of Pseudosciaena crocea, can effectively improve the breeding rate of excellent varieties of Pseudosciaena crocea, improve the germplasm of Pseudosciaena crocea, and improve the quality of fry. The provision of the DNA methylation molecular marker can provide valuable reference materials for the study of molecular marker assisted breeding of Pseudosciaena crocea and even other marine fish and the epigenetic regulation mechanism.

[0085] The method for screening the DNA methylation molecular marker of Pseudosciaena crocea provided by the present application comprises the following steps:

[0086] Step 1: The experimental material is a new strain of Pseudosciaena crocea "Fu Fa No. 1" from Ningde Fu Fa Aquatic Products Co., Ltd., and the Pseudosciaena crocea is cultured according to the Pseudosciaena crocea fry breeding technical specification of Ningde Fu Fa Aquatic Products Co., Ltd.; 314 tails of new strain F4 generation fry of about 13 months old are randomly selected in the same net cage under the same culture mode, each fish is well measured and marked, and a rice-sized muscle is cut and preserved in anhydrous ethanol. The 40 tails of high-value individuals with the largest body weight are marked as the largest individual group (L group), and the 40 tails of low-value individuals with the smallest body weight are marked as the smallest individual group (S group);

[0087] Step 2: Whole genome bisulfite sequencing (WGBS) is performed on the largest individual group and the smallest individual group to obtain a DNA methylation genetic map at the whole genome level, and differentially methylated regions (DMRs) of methylation C sites of three methylation modes (CG type, CHG type and CHH type) are found at the same position of the genomes of the L group and the S group, and the differentially methylated regions of the two groups are screened to obtain growth axis functional genes with differential methylation;

[0088] Among them, the identification of DMRs needs to have the following related characteristics:

[0089] (1) There are more than 5 methylated C bases in the region in at least one sample;

[0090] (2) The total sequencing depth of each methylation C site should be more than 10, and the supporting sequencing depth of the methylation C site should be more than 4;

[0091] (3) The length of the region should be between 40 bp and 10 kb;

[0092] (4) The distance between two adjacent methylation C sites should be no more than 200 bp;

[0093] (5) There is more than 2 times of average methylation level;

[0094] (6) Chi-square test P≤0.05;

[0095] Step 3, screening the growth-related genes with significant methylation difference in the upstream 2k and gene body region from the high-throughput sequencing results, designing qRT-PCR primers (SEQ ID No. 13-SEQ ID No. 20), detecting the expression level of these genes in different phenotype trait populations in the same family by qRT-PCR technology, detecting the correlation between the mRNA expression of the growth axis functional gene with differential methylation and methylation, and screening 4 genes (fgf11, fgfrs2, egflp7 and pdgfrα) with significant methylation in the promoter region (upstream 2k) and qRT-PCR difference from the sequencing results;

[0096] Step 4, Richer Company breeds 14-month-old seedlings of the same family, randomly selects 258 from them, takes 30 with the maximum weight and 35 with the minimum weight as the materials for verifying the differential methylation genes;

[0097] Step 5, designing BSP-PCR primers (as shown in SEQ ID No. 5-SEQ ID No. 12), verifying the average methylation level of a single gene in different phenotype trait individuals, obtaining the DNA methylation site with significant regulatory effect on the expression level of the growth functional gene, and obtaining the DNA methylation molecular marker of large yellow croaker—molecular marker fgf11, molecular marker fgfrs2, molecular marker egflp7 and molecular marker pdgfrα.

[0098] From the above description, the beneficial effects of the present application are that the method for screening the molecular marker of DNA methylation of large yellow croaker provided by the present application carries out whole genome bisulfite sequencing on a large individual group and a small individual group, obtains DNA methylation genetic map at the whole genome level, screens DMRs of the two groups, obtains growth axis functional genes with differential methylation therefrom, detects the correlation between the mRNA expression of the genes and methylation by using qRT-PCR technology, verifies the correlation between the DNA methylation level of part of the differential methylation functional genes and growth traits, and obtains DNA methylation sites which have a significant regulatory effect on the expression level of growth functional genes.

[0099] The method is based on the genetic information of large yellow croaker genome, combines high-throughput RNA-Seq, genome resequencing and bisulfite sequencing technology, carries out difference characteristic research on the epigenetic of the large yellow croaker growth trait difference significant group, screens the differential methylation characteristics of the genes at the whole genome level, and applies the screened molecular marker to production practice to assist large yellow croaker breeding, and provides an important reference for molecular marker assisted breeding of large yellow croaker and epigenetic regulation mechanism.

[0100] Embodiment 1:

[0101] The method for screening the molecular marker of DNA methylation of large yellow croaker provided by the present application comprises the following steps:

[0102] Step 1, the experimental material is from the new strain of "Fu Fa No. 1" large yellow croaker of self-breeding of Ningde Fu Fa Aquatic Products Co., Ltd., and the large yellow croaker is cultivated according to the large yellow croaker fry breeding technical specification of Ningde Fu Fa Aquatic Products Co., Ltd.; about 314 tails of 13-month-old new strain F4 generation fry are randomly selected in the same net cage in the same culture mode, each fish is well measured and marked, and a rice grain size of muscle is cut and preserved in anhydrous ethanol. The 40 tails of high-value individuals with the largest weight are marked as "extreme large individual group (L group)", and the 40 tails of low-value individuals with the smallest weight are marked as "extreme small individual group (S group)".

[0103] Step 2, whole genome bisulfite sequencing is carried out on the extreme large individual group and the extreme small individual group, DNA methylation genetic map at the whole genome level is obtained, the differentially methylated regions (DMRs) of the methylation C sites of the three methylation modes (CG type, CHG type and CHH type) at the same position of the genomes of the L group and the S group are found, the differentially methylated regions of the two groups are screened, and growth axis functional genes with differential methylation are obtained therefrom;

[0104] Among them, the identification of DMRs needs to have the following related characteristics:

[0105] (1) At least in one sample, there are more than 5 methylated C bases in the region;

[0106] (2) The total sequencing depth of each methylated C site should be more than 10, and the supporting sequencing depth of the methylated C site should be more than 4;

[0107] (3) The length of the region should be between 40 bp and 10 kb;

[0108] (4) The distance between two adjacent methylated C sites should not be more than 200 bp;

[0109] (5) There is more than 2 times of average methylation level;

[0110] (6) Chi-square test P≤0.05.

[0111] Step 3, screening from high-throughput sequencing results to obtain growth-related genes with significant methylation difference in the upstream 2k and gene body region, designing qRT-PCR primers (shown in Table 2), detecting the expression level of these genes in different phenotype trait populations in the same family by qRT-PCR technology, detecting the correlation between the mRNA expression of growth axis functional genes with differential methylation, and screening 4 genes (fgf11, fgfrs2, egflp7 and pdgfrα) with significant methylation and qRT-PCR difference in the promoter region (upstream 2k) from the sequencing results.

[0112] Table 2

[0113]

[0114]

[0115]

[0116] Step 4, Rich Harvest Company breeds 14-month-old seedlings of the same family, randomly selects 258 from them, takes 30 with the maximum weight and 35 with the minimum weight as the materials for verifying the differential methylation genes.

[0117] Step 5, designing primers (SEQ ID No. 5-SEQ ID No. 12), verifying the average methylation level of single gene in different phenotype trait individuals, obtaining DNA methylation sites with significant regulation effect on the expression level of growth functional genes, and obtaining DNA methylation molecular markers of large yellow croaker—molecular marker fgf11, molecular marker fgfrs2, molecular marker egflp7 and molecular marker pdgfrα;

[0118] Specifically, step 5.1, genomic DNA bisulfite conversion and purification preservation:

[0119] The qualified genomic DNA was treated by Epitect DNA Bisulfite Conversion Kit (QIAGEN) with the following reagents: 1 μg genomic DNA, 85 μL Bisulfite solution, 35 μL DNA protect buffer, and 140 μL RNase-free water. The reaction was performed in a PCR machine with the following program: 95°C for 5 min, 60°C for 20 min, 95°C for 5 min, and 60°C for 30 min.

[0120] The converted DNA was further purified. The DNA was transferred to a clean sterile 1.5 mL centrifuge tube, 310 μL Buffer BL was added, mixed, and centrifuged at 3000 g for 15 s. 250 μL 95% ethanol was added, vortexed, and centrifuged at 3000 g for 15 s. The entire solution was transferred to a new centrifugal column, centrifuged at 8000 g for 1 min, and the lower liquid was discarded. 500 μL Buffer BW was added to the centrifugal column, centrifuged at 8000 g for 1 min, and the lower liquid was discarded. 500 μL Buffer BD was added again, and the column was placed at room temperature for 15 min, centrifuged at 8000 g for 1 min, and the lower liquid was discarded. 500 μL Buffer BW was added, centrifuged at 8000 g for 1 min, and the lower liquid was discarded. The above steps were repeated once. 250 μL 95% ethanol was added, centrifuged at 8000 g for 1 min, and the lower liquid was discarded. Then, the column was centrifuged at 12000 g for 1 min to completely remove the residual ethanol. The centrifugal column was transferred to a new sterile 1.5 mL centrifuge tube, 30 μL Buffer EB was added to the column, and the tube was centrifuged at 8000 g for 1 min. The liquid in the tube was the purified DNA, which was stored at -20°C for later use.

[0121] Step 5.2, gene-specific amplification:

[0122] According to the high-throughput sequencing of fgf11, fgfrs2, pdgfrα, egfg7 four gene promoter region (Upstream2k) with differential methylation region, the control reference genome obtains its corresponding gene sequence fragment, and the possible CpG island is predicted by using CpG IslandSearch program, and the gene specific primer is designed at both ends of the CpG island to amplify the specific fragment, and the PCR system is as follows: purified DNA 0.5 μL, 10×Taq Buffer 2 μL, dNTP 0.5 μL, Taq polymerase 0.5 μL, F-primer 0.5 μL, R-primer 0.5 μL, sterile H2O 15.5 μL, and placed in the following PCR program: 95℃ 10min, 40 cycles (94℃ 30s, 55℃ 30s, 72℃ 40s), 72℃ 10min.

[0123] Step 5.3, gel recovery of PCR product:

[0124] According to the operation instruction of DNA purification recovery kit (Shanghai Jierui Company), the gel recovery of the target fragment of PCR product is carried out, and the concentration is measured by using NanoDrop-1000 ultramicro ultraviolet spectrophotometer.

[0125] Step 5.4, connection with carrier:

[0126] According to the operation instruction of pMD19-T vector connection kit (TaKaRa), 2 μL of the gel purified target fragment, 4.5 μL of Ligation Solution I, 0.5 of PMD19-T vector and 3 μL of sterile water are added in 10 μL system, and the connection is carried out at 16℃ overnight.

[0127] Step 5.5, transformation of target fragment:

[0128] The connected product is transferred to a centrifuge tube containing 100 μL of DH5α competent cells, and is immediately placed on ice for 30 min after being shaken several times; then it is heated in a 42℃ water bath for 90 s, and then immediately placed back on ice for 2 min, 890 μL of LB medium is added, and the activated cells are cultured in a 37℃ shaker at 200 r / h for 1 h. 100 μL of the activated bacterial liquid is aspirated and uniformly coated on the LB solid medium added with ampicillin, and the culture plate is placed in a 37℃ incubator and cultured for 15 h.

[0129] Step 5.6, target fragment detection, inoculation culture and sequencing:

[0130] Ten single colonies were randomly picked from the culture plate and detected by gene-specific primers and M13 primers to determine whether the sequence of the target fragment existed. The colonies with positive PCR detection were put into LB liquid medium containing 1‰ for expansion culture, and sent to Shanghai Jeery Company for sequencing to verify the methylation level of single gene in different phenotypic traits.

[0131] Analysis of the results of Example 1:

[0132] The methylation level refers to the ratio of the number of methylation of three types of C bases (CG, CHG and CHH) covered in the genome to all methylated C bases, which can be expressed as:

[0133] Methylation level = (number of sequences covering mC / total number of effective covered sequences) x 100%.

[0134] 1. Whole genome average methylation level

[0135] The whole genome average methylation level reflects the overall characteristics of the methylation map of the genome of a particular species, and the results are shown in Table 3.

[0136] Table 3

[0137] Sample C(％) CG (%) CHG (%) CHH (%) L population 7.03 71.89 0.71 0.73 S population 7.04 72.01 0.68 0.7

[0138] It can be seen that the whole genome average methylation level of this species of P. major is mainly of the CG type.

[0139] 2. Whole genome methylation level distribution trend

[0140] 2.1 Distribution proportion of CG, CHG and CHH in methylated C bases

[0141] At the whole genome level, there is a large difference in the composition proportion of three types of methylated cytosine (mCG, mCHG and mCHH) in different species. Especially, the number of each type of methylated C base and the proportion it occupies in all mC sites is an important characteristic of the whole genome methylation map of the species, and the results are shown in Table 4.

[0142] Table 4

[0143] Total mCs mCG mCHG mCHH L population number 12769211 11900847 220327 648037 L population proportion (%) 100 93.2 1.73 5.07 S population number 12326383 11415822 231815 678746 S population proportion (%) 100 92.61 1.88 5.51

[0144] It can be seen that the proportion of CG type methylation is the highest, indicating that the genome methylation of P. major population mainly occurs at CG sites.

[0145] 2.2 Methylation level distribution in methylated CG, CHG and CHH

[0146] The distribution of methylation level of three types of C bases (CG, CHG and CHH) in different species is different, so the distribution of methylation level of each type of methylation (mCG, mCHG and mCHH) can reflect the DNA methylation characteristics of the species. The results are shown in Figure 1-2

[0147] It can be seen that the mCG sites in L population and S population are generally in a high methylation level state, that is, most of the CG methylation sites are in a 90%-100% methylation level state, while the change trend of CHG and CHH sites is relatively slow, most of which are between 20%-50%.

[0148] 2.3 Methylation distribution characteristics of different genomic regions

[0149] The methylation level of different genomic regions (Genebody, Upstream2k, Downstream2k, Exon, Intron, CDS, 5'UTR, 3'UTR) has different biological functions in the genome. The research results show that the number of CG / CHG / CHH three types of methylation in the gene body region of the genome of Pseudosciaena crocea is the most, followed by intron, and the number of methylation in 3'UTR is the least.

[0150] 2.4 DNA methylation level in different transcription elements of genome

[0151] All coding gene sequences are divided into three different transcription element regions of upstream2k, genebody, downstream2k, and the methylation levels of the three different transcription element regions are counted. The results are shown in Figure 3-4

[0152] 3 Analysis of differentially methylated regions

[0153] 3.1 Statistics of differentially methylated regions related genes

[0154] ​​Comparing the DMRs related genes obtained from the whole genome methylation sequencing information of S group and L group statistics, 2204 differential methylation genes related to CG / CHG / CHH type DMRs regions were obtained, including 1506 up-regulated genes and 698 down-regulated genes. Among the three types of CG / CHG / CHH, there were 2171 CG type differential methylation genes, including 1505 up-regulated genes and 666 down-regulated genes; 333 differential methylation genes were located in the Upstream 2k region, 1609 in the genebody region and 229 in the Downstream 2k region; 33 CHG type differential methylation genes were obtained, including 1 up-regulated gene and 32 down-regulated genes; 1 differential methylation gene was located in the Upstream 2k region, 31 in the genebody region and 1 in the Downstream 2k region. No differential methylation related genes were screened from CHH type DMRs.

[0155] 3.2 GO enrichment analysis of DMRs related genes

[0156] The CG / CHG type DMRs were respectively mapped to each sub-node of the GO database, and the number of genes in each node was calculated, so as to obtain the list of DMRs significantly enriched in a certain GO subject and the number statistics of each differential marker gene, and to describe and analyze according to the three subjects of biological process (BP), cell component (CC) and molecular function (MF).

[0157] 3.2.1 GO enrichment analysis of CG type DMRs related genes

[0158] A total of 1899 CG type DMRs (covering 333 differential expression genes) in the Upstream 2k region were mapped to the GO function sub-node (the same DMRs related genes may be mapped to multiple physiological processes in the GO sub-node, the same below), among which 970 DMRs related genes were accumulated and mapped to the category BP, including 4 DMRs related genes involved in growth process and 86 DMRs related genes involved in development process; 649 DMRs related genes were accumulated and mapped to the CC category, among which the number of DMRs related genes involved in cell and cell part was the largest, both being 135; 280 DMRs related genes were accumulated and mapped to the MF category, among which 138 DMRs related genes were involved in binding process and 1 DMRs related gene was involved in transcription factor activity-protein binding.

[0159] A total of 10335 CG-type DMRs (covering 1609 differentially expressed genes) were mapped to GO function subnodes, of which 5165 DMRs-related genes of BP category contained 45 and 444 DMRs-related genes involved in growth and development processes, respectively; 3626 DMRs-related genes of CC category contained the most DMRs-related genes of cell and cell part, which were 747 and 746, respectively; and 1208 DMRs-related genes of MF category contained the most DMRs-related genes of 791 binding processes and 1 DMRs-related gene of electron carrier activity.

[0160] A total of 1317 CG-type DMRs (covering 229 differentially expressed genes) in the downstream 2k region were mapped to GO function subnodes, of which the number of DMRs-related genes of BP category was the most, which was 641, the number of DMRs-related genes of CC category was 481, and the number of DMRs-related genes of MF category was the least, which was 195. In the BP category, the number of DMRs-related genes involved in growth and development processes was 5 and 46, respectively.

[0161] 3.2.2 GO enrichment analysis of CHG-type DMRs-related genes

[0162] There was only one CHG-type DMRs-related gene (Probable G-protein coupled receptor 19) in the Upstream 2k region, which was mapped to the biological regulation, membrane, and signal sensor activity subnodes of the BP, CC, and MF categories of GO function, respectively.

[0163] A total of 177 CHG-type DMRs (covering 31 differentially expressed genes) in the Genebody region were mapped to GO function subnodes, of which 83 DMRs-related genes of BP category contained 6 DMRs-related genes involved in development processes; 64 DMRs-related genes of CC category were mapped; and 30 DMRs-related genes of MF category were mapped.

[0164] The CHG-type DMRs of 1 differentially methylated gene in the Downstream 2k region were mapped to the BP, CC, and MF categories of GO enrichment diagram, which contained the biological regulation, binding, organ part, membrane, organelle, cell, and cell part subnodes, respectively.

[0165] 3.3 Pathway enrichment analysis of DMRs-related genes

[0166] We used the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway database to identify pathways significantly enriched with differentially expressed DMRs-related genes, in order to determine the biochemical metabolic and signal transduction pathways involved by these genes.

[0167] 3.3.1 Pathway enrichment analysis of CG-DMRs related genes in the Upstream2k region

[0168] As shown in Table 5, CG-type DMRs in the upstream2k region of differentially methylated genes from the S and L populations (S vs L) were annotated to the KEGG metabolic pathways. A total of 79 KEGG pathways and 175 DMRs were annotated to the six major KEGG metabolic pathways. Among them, the material metabolism pathway involved the most pathways (27), with 38 DMRs; while the DMRs involved in cellular processes were the most numerous (44), with 12 pathways; and the human disease pathway involved the fewest pathways and DMRs, with 3 and 9 respectively.

[0169] Table 5

[0170] KEGG main pathway KEGG sub-pathway gene number Number of differential unigenes Cellular process 12 44 Environmental information processing 16 47 Genetic information processing 8 13 Human disease 3 9 Metabolism 27 38 Organic system 13 24

[0171] Among the 79 KEGG pathways mentioned above, 5 metabolic pathways with significant enrichment (P < 0.05) were screened, mainly involved in endocytosis, cytokine-cytokine receptor interaction, regulation of the actin cytoskeleton, N-glycan biosynthesis, and focal adhesion. The differential methylation enrichment in the endocytosis pathway reached a highly significant level (P < 0.01); the differential methylation in the actin cytoskeleton regulation pathway reached a significant level (P < 0.05), and 8 differentially methylated genes, including epidermal growth factor (EGF), EGFR, and p21-activated kinase 1 (PAK1), were screened in this pathway.

[0172] 3.3.2 Pathway enrichment analysis of gene body region-related genes CG-DMRs

[0173] As shown in Table 6, CG-type DMRs in the genebody regions of differentially methylated genes from the S and L populations were annotated into the KEGG pathway metabolic pathway. A total of 136 KEGG pathways and 1056 DMRs were annotated into KEGG pathways across the six major metabolic pathways. Among them, the material metabolism pathway involved the most pathways (68 in total) and had 226 DMRs; while the environmental information processing pathway had the most DMRs (295) and had 17 pathways; the human disease pathway involved the fewest pathways and DMRs, with 4 and 65 respectively.

[0174] Table 6

[0175] KEGG main pathway KEGG sub-pathway gene number Number of differential unigenes Cellular process 14 245 Environmental information processing 17 295 Genetic information processing 17 73 Human disease 4 65 Metabolism 68 226 Organic system 16 152

[0176] From 1056 differentially methylated KEGG pathways, 17 significantly enriched metabolic pathways (P < 0.05) were screened, mainly involved in the Notch signaling pathway, focal adhesion, adhesion junctions, MAPK signaling pathway, and TGF-β signaling pathway. Among these, the P and Q values ​​for Notch signaling, focal adhesion, and adhesion junctions were both highly significant. The P values ​​for seven other signaling pathways, including the ErbB signaling pathway, the AGEs-RAGE signaling pathway in diabetic complications, and insulin resistance, were also highly significant. Within the MAPK signaling pathway, 41 growth-related genes, including fibroblast growth factor, fibroblast growth factor receptor 1, and growth factor receptor-binding protein 2, were identified as DMRs.

[0177] 3.3.3 Pathway enrichment analysis of Downstream2k region-related genes CG-DMRs

[0178] As shown in Table 7, CG-type DMRs in the downstream2k region of differentially methylated genes from the two populations (S vs L) were annotated into the KEGG pathway metabolic pathway, resulting in 77 KEGG pathways and 162 DMRs annotated into the six major KEGG metabolic pathways. Among them, the material metabolism pathway involved the most, with 24 pathways and 30 DMRs; the environmental information processing pathway had the most DMRs (38), with 13 pathways; while the human disease pathway involved the fewest pathways and DMRs, with 4 pathways and 14 DMRs, respectively.

[0179] Table 7

[0180] KEGG main pathway KEGG sub-pathway gene number Number of differential unigenes Cellular process 12 29 Environmental information processing 13 38 Genetic information processing 10 20 Human disease 4 14 Metabolism 24 30 Organic system 14 31

[0181] Among the 77 KEGG pathways identified, five metabolic pathways showed significant enrichment (P < 0.05), primarily involved in the Notch signaling pathway, Jak-STAT signaling pathway, protein processing in the endoplasmic reticulum, adipokine signaling pathway, and herpes simplex infection pathway. Within the Notch signaling pathway, DMRs (Distributed Metabolic Regulators) of five genes (Notch, DVL, CLS, etc.) were identified.

[0182] 3.4 Screening and quantitative PCR analysis of growth-related genes in CG-DMRs

[0183] 3.4.1 Screening of growth-related genes in CG-DMRs

[0184] Thirteen growth trait-related genes were initially screened from CG-type DMRs, as shown in Table 8. These genes were used for quantitative PCR to verify the differential expression levels of gene mRNA and to validate and analyze the results of whole-genome BS sequencing.

[0185] Table 8

[0186]

[0187]

[0188] 3.4.2 Relationship between qRT-PCR analysis results and methylation levels

[0189] The relationship between the relative mRNA expression levels and DNA methylation of the genes listed in Table 8 was analyzed using qRT-PCR. The results showed a positive correlation between DNA methylation levels in the genebody region and relative mRNA expression levels, while a negative correlation was found between DNA methylation levels in the upstream2k region and relative mRNA expression levels. These results indicate that whole-genome BS sequencing data is reliable and can accurately detect the relationship between methylation levels and gene expression levels of growth-related genes.

[0190] 3.5 Association analysis between single-gene methylation level and growth traits

[0191] 3.5.1 Screening for differentially methylated genes

[0192] Four genes were screened from the upstream 2k region: fibroblast growth factor 11-like (fgf11), fibroblast growth factor receptor substrate 2 (fgfrs2), epidermal growth factor-like protein 7 (egflp7), and platelet-derived growth factor receptor alpha (pdgfrα).

[0193] 3.5.2 Amplification of Differentially Methylated Genes

[0194] After sulfite conversion, the muscle DNA samples were amplified using gene-specific BSP primers (as shown in SEQ ID No. 5-SEQ ID No. 12) to obtain electrophoretic patterns. The detection results for the fgfrs2 and fgf11 genes in 65 samples are shown below. Figure 9-10 As shown, sample 65 was used as a pre-amplification detection BSP primer for electrophoresis. After the BSP primers passed the detection, BSP sequencing was performed on all 65 samples. Therefore, the electrophoresis image only includes the first 64 samples. The detection results for the pdgfrα gene and egflp7 in the 20 samples are shown below. Figure 11-12 As shown.

[0195] 3.5.3 Sequence information of gene methylation regions

[0196] Differentially methylated region sequences of four genes (fgf11, fgfrs2, egflp7, and pdgfrα) were obtained by gene cloning and sequencing. The results are shown in Table 1, SEQ ID No. 1-SEQ ID No. 4.

[0197] 3.5.3 Single-gene methylation sequencing results

[0198] The methylation level of the fgf11 gene in family S was significantly higher than that in family L. The average methylation frequency of the fgf11 gene in family S was 0.5333, significantly higher than that in family L (0.448). Figure 5 Consistent with the findings of whole-genome BS sequencing.

[0199] The methylation level of the fgfrs2 gene in family S was significantly higher than that in family L. The average methylation frequency of the fgfrs2 gene in family S was 0.5367, significantly higher than that in family L (0.4383). Figure 6 Consistent with the findings of whole-genome BS sequencing.

[0200] The methylation level of the egflp7 gene in family S was significantly higher than that in family L. The average methylation frequency of the egflp7 gene in family S was 0.2532, significantly higher than that in family L (0.1464). Figure 7 Consistent with the findings of whole-genome BS sequencing.

[0201] The methylation level of the pdgfrα gene in family S was significantly higher than that in family L. The average methylation frequency of the pdgfrα gene in family S was 0.4033, significantly higher than that in family L (0.2786). Figure 8 Consistent with the findings of whole-genome BS sequencing.

[0202] Example 2:

[0203] The application of molecular markers for DNA methylation in large yellow croaker breeding includes the following steps:

[0204] Step a: Extract DNA from the muscle tissue of large yellow croaker (using the DNA extraction kit from Shanghai Jierui Company), and after sulfite conversion, perform PCR amplification using the primers shown in Table 1 (SEQ ID No. 5-SEQ ID No. 12);

[0205] Step b: Detect the PCR amplification products. Referring to the molecular markers fgf11, fgfrs2, egflp7 and pdgfrα shown in Table 1 (SEQ ID No.1-SEQ ID No.4), select individuals with low average methylation frequencies of the fgf11, fgfrs2, egflp7 and pdgfrα genes as breeding parents.

[0206] In summary, the method for screening molecular markers of DNA methylation in large yellow croaker provided by this invention utilizes WGBS to detect DNA methylation characteristics in large yellow croaker. Genes related to differentially methylated regions are screened from the methylation maps of large yellow croaker populations L and S. Thirteen growth-related genes were screened from CG-type DMRs-related genes, and the relationship between qRT-PCR and their methylation frequency was analyzed. Furthermore, from seven DMRs-related genes in the promoter region, fibroblast growth factor 11 (fgf11), fibroblast growth factor receptor substrate 2 (fgfrs2), epidermal growth factor-like protein 7 (egflp7), and platelet-derived growth factor receptor α (pdgfra) were screened. The methylation frequency of these four cell growth factor genes was higher in the S population than in the L population, and was negatively correlated with the mRNA expression level of these genes. Methylation in the promoter regions of these genes can directly regulate gene expression, indicating that these four genes play an important regulatory role in cell growth, proliferation, and individual growth and development in large yellow croaker.

[0207] The molecular markers obtained from the screening were applied to production practice to assist in the breeding of large yellow croaker, providing an important reference for molecular marker-assisted breeding and epigenetic regulatory mechanisms of large yellow croaker;

[0208] The molecular marker for DNA methylation in large yellow croaker provided by this invention can be applied to auxiliary breeding research in production practice; its application in large yellow croaker breeding, for screening of large yellow croaker broodstock, can effectively improve the breeding rate of superior varieties of large yellow croaker, improve the germplasm of large yellow croaker, and improve the quality of fish fry.

[0209] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent modifications made based on the content of the present invention specification and drawings, or direct or indirect applications in related technical fields, are similarly included within the patent protection scope of the present invention. sequence list <110> Ningde Normal University <120> Methods and qRT-PCR primers for screening molecular markers for DNA methylation in large yellow croaker <160> 20 <170> SIPOSequenceListing 1.0 <210> 1 <211> 292 <212> DNA <213> Artificial sequence <400> 1 tgtctaacac acacacacat ggacacacac tgtgcagctg gactcaacag aaccagaccg 60 gactactgtc acccaccagt ggagactctc gcatgagcag cggcaacaca gcgtctaccg 120 taaggcatgg ctacctgcca gcacccccac ccgccccctc ccctattccc cttcacctcc 180 tctcctctcc ctccctctct gtctccttcc attcattcat ccctcgttcc cccagctgtg 240 [[ID=⑧]]aggcggcatg cgtggctgaa ggacacagcg ctgcgctctg ctgtggtgta gc 292[[ID=⑨]] [[ID=⑩]]<210> 2[[ID=⑪]] [[ID=⑫]]<211> 477[[ID=⑬]] [[ID=⑭]]<212> DNA[[ID=⑮]] [[ID=⑯]]<213> Artificial sequence[[ID=⑰]] [[ID=⑱]]<400> 2[[ID=⑲]] [[ID=⑳]]cggggcttca ccacattgga actctcggaa gttcagccaa gttgccgaga tttagccgca 60[[ID=㉑]] [[ID=㉒]]gctagcagca gagacgctcc gaccgcagca aacttggttt tcatcggata aaaagtgcgt 120[[ID=㉓]] [[ID=㉔]]ttggacggaa gctcgtcccc cacagtgacc cacggaagat tgtcgatgga tttaaaccga 180[[ID=㉕]] [[ID=㉖]]tgttcgctgt gtgatcggcg cagacggcag cggtagagtc tttgagagaa gttgtgattg 240[[ID=㉗]] [[ID=㉘]]ttgtttttgg cgctctagca gcgcagagga aagatggagg ctgcgctggg ttagcacaga 300[[ID=㉙]] [[ID=㉚]]gctaacgcta gctgttggag gactggccag cgtcgtttcg ggacgaaccg agctgctcca 360[[ID=㉛]] It should be noted that there seems to be an incorrect "⑧" in the translation of line 8. It should probably be "". Also, the "⑩", "⑪", etc. are just numbered for clarity in showing the correspondence with the original tags in the translation process. The original text seems to be some DNA sequences with related metadata tags.cacctggacg ggctccggga cgtttggacg ctgcatttga acacggtgac ggttactcgt 420 aagtatggct ccatgacttc acggctgttt tcacggcggc tcgcagcgga atgcgtc 477 <210> 3 <211> 585 <212> DNA <213> Artificial Sequence <400> 3 tcgccagtcc actcacaggg agcaccaaga acttaaaccg agaggttttt tactatttca 60 accctcgaac cgaacggtca acttttaaaa aagaagttgg caccatggaa gactcctctt 120 atccaccggt ccggcggctc gtgcccatgg cggccgctca gcctgcgcgc gcacacgcac 180 ctgatgaccc cagactgcga cggtaagatt tatttacata ttaataatta ttactcataa 240 acatgttcag acatgaggaa ccatgtacaa ctgtgtgtgt tttagattta tttatagtgt 300 ttttttatat aataaaatat taaataaggt cagactgtac tttacgcaga gaaagaatgc 360 agacatacag ttaaacaaag ctaataaaca aatgtaagca tataatggag atgtattata 420 tacaggacaa taatttatgc aactacaggt gagattctac ctggtttaca cgcatgtatt 480 gaggttatat gtatagcaca gacaaatata tgtgacaaga aaaccaagga cctctatgta 540 tggcacttta ttccctttaa caagaccac tgagtgattt ccata <210> 4 <211> 579 <212> DNA <213> The snowstorm <400> 4 ctctccagtc cacttcaggg aggtccacgt tcttgaaaaa caaaaacgaa agtatagatg gactgatgat aaatgtatag aagacagat agacaggtga atgggtggac agagtcggga 180. ctttggctga gggacaaacg ctgccgaccc tgttaaagac acacttcagc acgctggaca gctccttcag ggacctccgc cccaaggcca cagacagaca cagacaga cagacagaca 240 300. gacacacat gtatgtgtct acaagacgag ctgttgtatg tccaacacgt gtacttaggt ctggtatatt ccaggtactc ttaatcataa agtcagacgt tggaaaaatc gttgacatga 420. 420. 420. 420. 420. 420. 420. 420. 420. 420. 420 acgtttattt tatttgtatt atttaagaag ttgacagaga aacgtaaact gacgtttttt agacgctgtc tcacgtcagg ttgttgttat tctcggatgt acggccttgt ttagacacag 540 ggcggaaacg tgtcgagatg atgtgcaatg gatgtatgt 579 <210> 5 <211> 27 <212> DNA <213> Artificial sequence <400> 5 tgtgcagctg gactcaacag aaccaga 27 <210> 6 <211> 18 <212> DNA <213> Artificial sequence <400> 6 cgctgtgtcc ttcagcca 18 <210> 7 <211> twenty four <212> DNA <213> Artificial sequence <400> 7 acattggaac tctcggaagt tcag 24 <210> 8 <211> twenty three <212> DNA <213> Artificial sequence <400> 8 gaaaacagcc gtgaagtcat gga 23 <210> 9 <211> twenty three <212> DNA <213> Artificial sequence <400> 9 aaaagaagtt ggcaccatgg aag 23 <210> 10 <211> 27 <212> DNA <213> Artificial sequence <400> 10 agtgccatac atagaggtcc ttggttt 27 <210> 11 <211> twenty one <212> DNA <213> Artificial sequence <400> 11 gggactttgg ctgagggaca a 21 <210> 12 <211> 25 <212> DNA <213> Artificial sequence <400> 12 gagacagcgt ctaaaaaacg tcagt 25 <210> 13 <211> 20 <212> DNA <213> Artificial sequence <400> 13 ctcccatttg tctccgctgt 20 <210> 14 <211> 20 <212> DNA <213> Artificial sequence <400> 14 gtctcagata gacggccagc 20 <210> 15 <211> twenty two <212> DNA <213> Artificial sequence <400> 15 catcttcaac ttcgacttcc gc 22 <210> 16 <211> twenty two <212> DNA <213> Artificial sequence <400> 16 gttaccggca ttgttgtcca tc 22 <210> 17 <211> 20 <212> DNA <213> Artificial sequence <400> 17 tgcagcacat acaagaccgt 20 <210> 18 <211> 20 <212> DNA <213> Artificial sequence <400> 18 ggcttggttg cagttgtgag 20 <210> 19 <211> twenty two <212> DNA <213> Artificial sequence <400> 19 agccctgatg tcagaactga ag 22 <210> 20 <211> twenty two <212> DNA <213> Artificial sequence <400> 20 ctggattcag gctgaggaag tt 22

Claims

1. A method for screening DNA methylation molecular markers of Pseudosciaena crocea, characterized in that, It comprises the following steps: Step 1, screening the extremely large individual group and the extremely small individual group with significant difference in growth traits from the breeding population under the same breeding condition; Step 2, obtaining the DNA methylation genetic map at the whole genome level by performing whole genome bisulfite sequencing on the extremely large individual group and the extremely small individual group, and screening the differential methylation region of the two groups to obtain the growth axis functional genes with differential methylation; Step 3, designing qRT-PCR primers and detecting the correlation between the mRNA expression of the growth axis functional genes with differential methylation and methylation by using qRT-PCR technology to screen the differential methylation functional genes; Step 4, verifying the correlation between the single gene DNA methylation level and growth traits of the differential methylation functional genes by using the primers for detecting the DNA methylation molecular marker of large yellow croaker to obtain the DNA methylation site which has a significant regulatory effect on the expression level of the growth functional genes and obtain the DNA methylation molecular marker of large yellow croaker; The DNA methylation molecular marker of large yellow croaker comprises a molecular marker fgf11, a molecular marker fgfrs2, a molecular marker egflp7 and a molecular marker pdgfrα; The nucleotide sequence of the molecular marker fgf11 is SEQ ID No. 1; The nucleotide sequence of the molecular marker fgfrs2 is SEQ ID No. 2; The nucleotide sequence of the molecular marker egflp7 is SEQ ID No. 3; The nucleotide sequence of the molecular marker pdgfrα is SEQ ID No. 4; The primers for detecting the DNA methylation molecular marker of large yellow croaker comprise: The fgf11 upstream primer with the nucleotide sequence of SEQ ID No. 5; The fgf11 downstream primer with the nucleotide sequence of SEQ ID No. 6; The fgfrs2 upstream primer with the nucleotide sequence of SEQ ID No. 7; The fgfrs2 downstream primer with the nucleotide sequence of SEQ ID No. 8; The egflp7 upstream primer with the nucleotide sequence of SEQ ID No. 9; The egflp7 downstream primer with the nucleotide sequence of SEQ ID No. 10; The pdgfrα upstream primer with the nucleotide sequence of SEQ ID No. 11; The pdgfrα downstream primer with the nucleotide sequence of SEQ ID No. 12; The DNA methylation refers to the process of covalently transferring the methyl group on the S-adenosyl methionine molecule to the 5th carbon atom of cytosine CpG dinucleotide by the catalytic action of DNA methylation transferase to form 5-methylcytosine; The differential methylation region in step 1 is the differential methylation region of the methylation C site, and the differential methylation region of the methylation C site should have the following characteristics: There are more than 5 methylation C bases in the region in at least one sample; The total sequencing depth of each methylation C site should be more than 10, and the support sequencing depth of the methylation C site should be more than 4; The length of the region should be between 40 bp and 10 kb; The distance between two adjacent methylated C sites is not more than 200bp; The average methylation level is more than 2 times; Chi-square test P≤0.

05.

2. The method for screening the molecular marker of DNA methylation of Pseudosciaena crocea according to claim 1, characterized in that, The qRT-PCR primers in step 3 include: The fgf11 upstream primer with the nucleotide sequence of SEQ ID No. 13; The fgf11 downstream primer with the nucleotide sequence of SEQ ID No. 14; The fgfrs2 upstream primer with the nucleotide sequence of SEQ ID No. 15; The fgfrs2 downstream primer with the nucleotide sequence of SEQ ID No. 16; The egflp7 upstream primer with the nucleotide sequence of SEQ ID No. 17; The egflp7 downstream primer with the nucleotide sequence of SEQ ID No. 18; The pdgfrα upstream primer with the nucleotide sequence of SEQ ID No. 19; The pdgfrα downstream primer with the nucleotide sequence of SEQ ID No.

20.

3. qRT-PCR primers for screening DNA methylation molecular markers of Larimichthys crocea, characterized by, The qRT-PCR primers in step 3 include: ​ The fgf11 upstream primer with the nucleotide sequence of SEQ ID No. 13; The fgf11 downstream primer with the nucleotide sequence of SEQ ID No. 14; The fgfrs2 upstream primer with the nucleotide sequence of SEQ ID No. 15; The fgfrs2 downstream primer with the nucleotide sequence of SEQ ID No. 16; The egflp7 upstream primer with the nucleotide sequence of SEQ ID No. 17; The egflp7 downstream primer with the nucleotide sequence of SEQ ID No. 18; The pdgfrα upstream primer with the nucleotide sequence of SEQ ID No. 19; The pdgfrα downstream primer with the nucleotide sequence of SEQ ID No.

20. The DNA methylation molecular markers of the big yellow croaker include the molecular marker fgf11, the molecular marker fgfrs2, the molecular marker egflp7 and the molecular marker pdgfrα; The nucleotide sequence of the molecular marker fgf11 is SEQ ID No. 1; The nucleotide sequence of the molecular marker fgfrs2 is SEQ ID No. 2; The nucleotide sequence of the molecular marker egflp7 is SEQ ID No. 3; The nucleotide sequence of the molecular marker pdgfrα is SEQ ID No. 4; The DNA methylation refers to the process of covalently transferring the methyl on the S-adenosyl methionine molecule to the 5th carbon atom of the cytosine CpG dinucleotide by the catalysis of the DNA methylation transferase to form 5-methylcytosine.

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