CN113337617A

CN113337617A - Large yellow croaker DNA methylation molecular marker and application thereof in breeding

Info

Publication number: CN113337617A
Application number: CN202110724168.7A
Authority: CN
Inventors: 韩坤煌; 王艺磊; 张子平; 刘伟; 阮少江; 黄伟卿; 李菲艳; 陈欣欣
Original assignee: Ningde Normal University
Current assignee: Ningde Normal University
Priority date: 2021-06-29
Filing date: 2021-06-29
Publication date: 2021-09-03
Anticipated expiration: 2041-06-29
Also published as: CN114891897A; CN113337617B

Abstract

The invention discloses a large yellow croaker DNA methylation molecular marker, a screening method of the molecular marker and application of the molecular marker in large yellow croaker breeding; the molecular markers of the DNA methylation of the large yellow croaker comprise a molecular marker fgf11, a molecular marker fgfrs2, a molecular marker egflp7 and a molecular marker pdgfr alpha. The method for screening the molecular marker of the DNA methylation of the large yellow croaker, provided by the invention, is used for carrying out the research on the difference characteristics of a large yellow croaker population with obvious growth character difference in epigenetics by combining high-throughput RNA-Seq, genome re-sequencing and bisulfite sequencing technologies on the basis of the genetic information of the large yellow croaker genome and screening the difference methylation characteristics of the gene on the whole genome level; the large yellow croaker DNA methylation molecular marker provided by the invention can be applied to large yellow croaker breeding, assists in breeding research and can effectively improve the breeding rate of a good large yellow croaker variety.

Description

Large yellow croaker DNA methylation molecular marker and application thereof in breeding

Technical Field

The invention belongs to the technical field of molecular markers, and particularly relates to a large yellow croaker DNA methylation molecular marker and application thereof in breeding.

Background

The large yellow croaker (Larimichthyscocea) is a special seawater economic cultured fish in China, the culture and seedling yield of the large yellow croaker is the first seawater fish in China, and the large yellow croaker occupies an important position in the seawater culture industry in China. At present, the large yellow croaker industry enters a transformation and upgrading stage, and the industry has increasingly strong expectations for excellent varieties (strains) with fast growth, strong stress resistance and high quality. Therefore, the breeding of the large yellow croaker becomes an important link in the development of the large yellow croaker industry. The molecular breeding technology is a modern biotechnology breeding means emerging and developed for overcoming the problems of low breeding efficiency, long period and the like of the traditional breeding technology, and is widely applied to fish breeding. Such as DongDongjie, successfully cultivates a new carp variety of Cyprinus carpio (Cyprinus carpio) by using PIT marking technology and BLUP method; li Shengjie, etc. utilizes molecular biology technology to combine with traditional breeding technology to breed a new variety of Micropterus salmoides (Lac Regis Apis) 1 with obvious growth advantage; the great wallichow fingerlings (Changfeng silver carp) which is a new variety with fast growth and good body shape is successfully cultivated by comprehensively utilizing gynogenesis, molecular marking and population breeding technologies; the novel Carassius auratus gibelio variety 'Zhongke No. 5' cultivated by units such as Chinese academy of sciences hydrophyte is cultivated by combining a population breeding technology, a gynogenesis technology and an SSR molecular marker assisted breeding technology, and has the characteristics of remarkable growth advantage and strong virus and myxosporidium resistance. The research results show that the molecular marker assisted breeding is used as a modern biotechnology breeding means, and the development potential and the application prospect are very huge.

Epigenetics (Epigenetics) is one of the research hotspots in the field of molecular biology in recent years. Unlike classical genetics theory, in which inheritance of a phenotypic characteristic is mainly determined by a DNA base sequence, epigenetic regulation refers to the phenomenon that expression variation of a gene is stably inherited without substantial change of the DNA sequence, so that the phenotypic characteristic is changed and is potentially reversible, and the relationship between a genotype and the phenotypic characteristic is reflected. Therefore, the method can lay a foundation for selecting the large yellow croaker of an excellent variety (strain) by researching the epigenetic regulation mechanism of the large yellow croaker. However, the current research on molecular regulation mechanism and genetic basis related to growth traits of large yellow croakers is not sufficient, and the transcriptome and epigenetic regulation mechanism on the whole level of the growth axis of the large yellow croakers is not reported yet.

Epigenetic-related regulatory mechanisms mainly include the following three aspects: DNA methylation, noncoding RNA interactions, and histone modification. The DNA methylation refers to a process of covalently transferring a methyl group on an S-adenosylmethionine molecule to a carbon atom at the 5 th position of cytosine CpG dinucleotide by the catalytic action of DNA methylation transferase to form 5-methylcytosine (5-mC), and is one of important modification modes of epigenetic regulation. Methylation modification of specific gene regions of genome DNA can have important influence on aspects such as chromosome conformation, gene expression regulation, genome defense, genome imprinting and the like, CpG methylation located in a promoter region and an enhancer region is in negative correlation with gene transcription activity, and high methylation in a gene main body region is in positive correlation with gene expression; meanwhile, organisms can form methylation types with different degrees according to the change of environment, then different phenotypes are generated to adapt to new environment and can be inherited to the next generation, and the methylation of DNA is an important method for researching the difference of species growth traits.

Therefore, the research on the difference characteristics of the large yellow croaker population with obvious growth character difference on transcriptome, genome and epigenetics is carried out according to the genetic information of the large yellow croaker genome, and the obtained molecular marker which can be applied to the breeding of the large yellow croaker and further can obtain the DNA methylation of a good variety (strain) with good genetic character becomes a technical problem to be solved at present.

Disclosure of Invention

In order to overcome the defects of the prior art, the technical problems to be solved by the invention are as follows: provides a DNA methylation molecular marker which can be applied to breeding of excellent large yellow croaker varieties (strains) and a screening method thereof.

In order to solve the technical problems, the invention adopts a technical scheme that: the molecular markers of DNA methylation of the large yellow croaker comprise molecular marker fgf11, molecular marker fgfrs2, molecular marker egflp7 and molecular marker pdgfr alpha;

the nucleotide sequence of the molecular marker fgf11 is shown as SEQ ID No. 1; the nucleotide sequence of the molecular marker fgfrs2 is shown in SEQ ID No. 2; the nucleotide sequence of the molecular marker egflp7 is shown as SEQ ID No. 3; the nucleotide sequence of the molecular marker pdgfr alpha is shown as SEQ ID No. 4.

The invention adopts a technical scheme that: the method for screening the molecular marker for DNA methylation of the large yellow croaker comprises the following steps:

step 1, screening a maximum individual group and a minimum individual group with obvious differences in growth traits from breeding populations under the same breeding conditions;

step 2, carrying out whole genome bisulfite sequencing on the extremely large individual group and the extremely small individual group to obtain DNA methylation genetic maps on the whole genome level, screening to obtain differential methylation regions of the two groups, and obtaining growth axis functional genes with differential methylation from the differential methylation regions;

step 3, designing qRT-PCR primers, detecting the correlation between the mRNA expression condition and methylation of the growth axis functional genes with differential methylation by utilizing a qRT-PCR technology, and screening the differential methylation functional genes;

and 4, adopting the primers of SEQ ID No.5-SEQ ID No12 to verify the correlation between the single-gene DNA methylation level and the growth traits of the differential methylation functional genes, obtaining DNA methylation sites with obvious regulation and control effects on the expression level of the growth functional genes, and obtaining the molecular marker of the DNA methylation of the large yellow croakers.

The invention adopts a technical scheme that: the application of the molecular marker for DNA methylation of the large yellow croaker in breeding of the large yellow croaker comprises the following steps:

step a, extracting DNA (DNA extraction kit of Shanghai Czeri company) from muscle tissues of large yellow croakers, converting the DNA with sulfite, and performing PCR amplification by using primers of SEQ ID No.5-SEQ ID No 12;

and b, detecting the PCR amplification product, and selecting an individual with low average methylation frequency of fgf11 gene, fgfrs2 gene, egflp7 gene and pdgfr alpha gene as a breeding parent by referring to the molecular marker fgf11, the molecular marker fgfrs2, the molecular marker egflp7 and the molecular marker pdgfr alpha.

The invention has the beneficial effects that: the large yellow croaker DNA methylation molecular marker provided by the invention can be applied to production practice for auxiliary breeding research; the method can be applied to breeding of the large yellow croaker, is used for screening parent large yellow croakers, and can effectively improve the breeding speed of excellent large yellow croakers, improve the germplasm of the large yellow croakers and improve the quality of fry; the method for screening the molecular marker of the DNA methylation of the large yellow croaker, provided by the invention, is used for developing the research on the difference characteristics of a large yellow croaker population with obvious growth character difference in epigenetics by combining high-throughput RNA-Seq, genome re-sequencing and bisulfite sequencing technologies on the basis of the genetic information of the large yellow croaker genome, screening the difference methylation characteristics of genes on the whole genome level of the large yellow croaker population, applying the screened molecular marker to production practice to assist the breeding of the large yellow croaker, and providing important reference for the molecular marker-assisted breeding and the epigenetics regulation mechanism of the large yellow croaker.

Drawings

FIG. 1 is a graph showing the DNA methylation levels in different transcription elements of the L population of example 1 in accordance with an embodiment of the present invention;

FIG. 2 is a graph showing the DNA methylation levels in different transcription elements of the L population of example 1 in accordance with an embodiment of the present invention;

FIG. 3 shows the result of verifying the methylation level of fgf11 gene in example 1 according to an embodiment of the present invention;

FIG. 4 shows the results of verifying the methylation level of the fgfrs2 gene of example 1 according to an embodiment of the present invention;

FIG. 5 shows the results of verifying the methylation level of the egflp7 gene of example 1 according to an embodiment of the present invention;

FIG. 6 shows the result of verifying the methylation level of pdgfr alpha gene of example 1 according to an embodiment of the present invention;

FIG. 7 shows the results of the amplification of the fgfrs2 gene of example 1 according to an embodiment of the present invention;

FIG. 8 is a view showing the fgf11 gene amplification result of example 1 according to the embodiment of the present invention;

FIG. 9 shows the result of pdgfr. alpha. gene amplification in example 1 according to an embodiment of the present invention;

FIG. 10 shows the result of amplification of the egflp7 gene of example 1, which is an embodiment of the present invention.

Detailed Description

In order to explain technical contents, achieved objects, and effects of the present invention in detail, the following description is made with reference to the accompanying drawings in combination with the embodiments.

The large yellow croaker DNA methylation molecular markers are shown in table 1 and comprise molecular markers fgf11, fgfrs2, egflp7 and pdgfr alpha;

the nucleotide sequences of the molecular marker fgf11, the molecular marker fgfrs2, the molecular marker egflp7 and the molecular marker pdgfr alpha are respectively shown as SEQ ID No.1-SEQ ID No. 4.

Further, the primers for detecting the above-mentioned molecular markers for DNA methylation of large yellow croaker, as shown in table 1, include: fgf11 upstream primer F1 with the nucleotide sequence shown as SEQ ID No. 5;

fgf11 downstream primer R1 with the nucleotide sequence shown as SEQ ID No. 6;

an upstream primer F1 of fgfrs2 with the nucleotide sequence shown as SEQ ID No. 7;

a downstream primer R1 of fgfrs2 with the nucleotide sequence shown as SEQ ID No. 8;

an upstream primer F1 of egflp7 with the nucleotide sequence shown as SEQ ID No. 9;

the downstream primer R1 of egflp7 with the nucleotide sequence shown as SEQ ID No. 10;

a pdgfr alpha upstream primer F1 having the nucleotide sequence shown in SEQ ID No. 11;

the downstream primer R1 of pdgfr alpha with the nucleotide sequence shown in SEQ ID No. 12.

Further, the square primers used for screening the above-mentioned molecular markers for DNA methylation of large yellow croaker, see table 1, include: fgf11 upstream primer F2 with the nucleotide sequence shown as SEQ ID No. 13;

fgf11 downstream primer R2 with the nucleotide sequence shown as SEQ ID No. 14;

an upstream primer F2 of fgfrs2 with the nucleotide sequence shown as SEQ ID No. 15;

a downstream primer R2 of fgfrs2 with the nucleotide sequence shown as SEQ ID No. 16;

an upstream primer F2 of egflp7 with the nucleotide sequence shown as SEQ ID No. 17;

the downstream primer R2 of egflp7 with the nucleotide sequence shown as SEQ ID No. 18;

a pdgfr alpha upstream primer F2 having the nucleotide sequence shown in SEQ ID No. 19;

the downstream primer R2 of pdgfr alpha with the nucleotide sequence shown in SEQ ID No. 20.

TABLE 1

From the above description, the beneficial effects of the present invention are: the large yellow croaker DNA methylation molecular marker provided by the invention can be applied to auxiliary breeding research in production practice, can be used for screening parent large yellow croakers, and can effectively improve the breeding speed of excellent large yellow croakers, improve the germplasm of the large yellow croakers and improve the quality of fry. The DNA methylation molecular marker can provide valuable reference data for researching molecular marker assisted breeding and epigenetic regulation and control mechanisms of the large yellow croaker and other seawater fishes.

The method for screening the molecular marker for DNA methylation of the large yellow croaker comprises the following steps:

step 1, breeding large yellow croakers according to the large yellow croaker fry breeding technical specification of the Ningde Rich water-producing company, wherein experimental materials are from a self-breeding new large yellow croaker strain of the Rich water-producing company Limited in Ningde city; randomly extracting 314 tails of the new strain F4 generation fries of about 13 months old from the same net cage in the same culture mode, carrying out biological measurement and marking on each fish, shearing the muscle with the size of rice grains, and storing the muscle in absolute ethyl alcohol. Marking the 40-tailed high-value individual group with the largest weight as a maximum individual group (L group), and marking the 40-tailed low-value individual group with the smallest weight as a minimum individual group (S group);

step 2, performing genome bisulfite sequencing (WGBS) on the extremely large individual group and the extremely small individual group to obtain a DNA methylation genetic map on the whole genome level, searching Differential Methylation Regions (DMRs) of methylation C sites of three methylation patterns (CG type, CHG type and CHH type) at the same position of two sample genomes of the L group and the S group, screening to obtain the differential methylation regions of the two groups, and obtaining a growth axis functional gene with differential methylation from the differential methylation regions; among them, the identification of DMRs needs to have the following characteristics:

(1) more than 5 methylated C bases are present in the region in at least one sample; (2) the requirement of the total sequencing depth of each methylated C site is more than 10, and the supported sequencing depth of each methylated C site is more than 4; (3) the length of the region should be between 40bp and 10 kb; (4) the distance between two adjacent methylation C sites is not more than 200 bp; (5) there is an average methylation level of more than 2 times; (6) checking that P is less than or equal to 0.05 by chi fang;

step 3, screening growth related genes with significant methylation differences in the upstream2k and gene body regions from a high-throughput sequencing result, designing qRT-PCR primers (SEQ ID No.13-SEQ ID No.20), detecting the expression levels of the genes in different phenotypic character populations in the same family by using a qRT-PCR technology, detecting the correlation between the mRNA expression condition and the methylation of growth axis functional genes with differential methylation, and screening 4 genes (fgf11, fgfrs2, egflp7 and pdgfr alpha) with significant methylation and qRT-PCR differences in a promoter region (upstream 2k) from a sequencing result;

step 4, cultivating 14-month-old same family seedlings by a rich development company, randomly selecting 258 tails from the family seedlings, and taking the maximum value 30 tails and the minimum value 35 tails of the body weight as materials for verifying differential methylation genes;

and 5, designing a BSP-PCR primer (shown as SEQ ID No.5-SEQ ID No.12), verifying the average methylation level of a single gene in individuals with different phenotypic characters, obtaining a DNA methylation site with a remarkable regulation and control effect on the expression level of a growth functional gene, and obtaining molecular markers of DNA methylation of the large yellow croakers, namely molecular marker fgf11, molecular marker fgfrs2, molecular marker egflp7 and molecular marker pdgfr alpha.

From the above description, the beneficial effects of the present invention are: the method for screening the molecular marker of the DNA methylation of the large yellow croaker, provided by the invention, comprises the steps of carrying out whole genome bisulfite sequencing on a very large individual group and a very small individual group to obtain a DNA methylation genetic map on the whole genome level, screening to obtain DMRs of the two groups, obtaining growth axis functional genes with differential methylation from the DMRs, detecting the correlation between the mRNA expression conditions and the methylation of the genes by utilizing a qRT-PCR technology, screening partial differential methylation functional genes to carry out verification on the correlation between the single-gene DNA methylation level and the growth traits, and obtaining DNA methylation sites with obvious regulation and control effects on the expression level of the growth functional genes;

on the basis of the genetic information of the pseudosciaena crocea genome, the method combines high-throughput RNA-Seq, genome re-sequencing and bisulfite sequencing technologies, develops the research on the difference characteristics of the pseudosciaena crocea growth character difference significant population on epigenetics, screens the difference methylation characteristics of genes on the whole genome level, applies the screened molecular marker to production practice to assist pseudosciaena crocea breeding, and provides important reference for molecular marker assisted breeding and epigenetics regulation and control mechanisms of the pseudosciaena crocea.

Example 1:

step 1, the experimental materials are from a self-breeding and self-breeding 'Rich-hair No. 1' large yellow croaker strain of the Ningde Rich-hair aquatic product Limited company, and large yellow croaker breeding is carried out according to the large yellow croaker fingerling breeding technical specification of the Ningde Rich-hair aquatic product company; randomly extracting 314 tails of the new strain F4 generation fries of about 13 months old from the same net cage in the same culture mode, carrying out biological measurement and marking on each fish, shearing the muscle with the size of rice grains, and storing the muscle in absolute ethyl alcohol. The 40-tailed high-value individual group with the largest weight is marked as the maximum individual group (L group), and the 40-tailed low-value individual group with the smallest weight is marked as the minimum individual group (S group).

2, carrying out whole genome bisulfite sequencing on the extremely large individual group and the extremely small individual group to obtain a DNA methylation genetic map on the whole genome level, searching differential methylation regions of methylation C sites of three methylation modes (CG type, CHG type and CHH type) at the same position of the genomes of the two samples of the L group and the S group, screening to obtain the differential methylation regions of the two groups, and obtaining a growth axis functional gene with differential methylation from the differential methylation regions;

among them, the identification of DMRs needs to have the following characteristics: (1) more than 5 methylated C bases are present in the region in at least one sample; (2) the requirement of the total sequencing depth of each methylated C site is more than 10, and the supported sequencing depth of each methylated C site is more than 4; (3) the length of the region should be between 40bp and 10 kb; (4) the distance between two adjacent methylation C sites is not more than 200 bp; (5) there is an average methylation level of more than 2 times; (6) the chi-square test P is less than or equal to 0.05.

And 3, screening growth related genes with significant methylation differences in the upstream2k and gene body regions from the high-throughput sequencing result, designing qRT-PCR primers (shown in table 2), detecting the expression levels of the genes in different phenotypic character populations in the same family by using the qRT-PCR technology, detecting the correlation between the mRNA expression condition and the methylation of growth axis functional genes with the differential methylation, and screening 4 genes (fgf11, fgfrs2, egflp7 and pdgfr alpha) with significant methylation and qRT-PCR differences in a promoter region (upstream 2k) from the sequencing result.

TABLE 2

And 4, cultivating 14-month-old same family seedlings by a rich-developing company, randomly selecting 258 tails from the seedlings, and taking the maximum value 30 tails and the minimum value 35 tails of the body weight as materials for verifying differential methylation genes.

Step 5, designing a primer (SEQ ID No.5-SEQ ID No.12), verifying the average methylation level of a single gene in individuals with different phenotypic traits, obtaining a DNA methylation site which has a remarkable regulation and control effect on the expression level of a growth functional gene, and obtaining molecular markers of DNA methylation of the large yellow croakers, namely molecular marker fgf11, molecular marker fgfrs2, molecular marker egflp7 and molecular marker pdgfr alpha;

specifically, step 5.1, genomic DNA bisulfite conversion and purification storage:

and (3) treating the qualified genomic DNA by using an Epitect DNA bisulfite rapid conversion kit (QIAGEN company), wherein the added reagent materials are as follows: adding RNase-free water into 1 mu g of genome DNA, 85 mu L of Bisulfite solution and 35 mu L of DNA protection buffer until the volume is 140 mu L, and placing the mixture into a PCR instrument to carry out transformation in a reaction program of 95 ℃ for 5min, 60 ℃ for 20min, 95 ℃ for 5min and 60 ℃ for 30 min;

the transformed DNA was further purified. Firstly, transferring DNA into a clean sterile 1.5mL centrifugal tube, adding 310 mu L Buffer BL, uniformly mixing, and centrifuging for 15s at 3000 g; adding 250 mu L of 95% ethanol, uniformly mixing by vortex, and centrifuging 3000g for 15 s; transferring all the solution into a new centrifugal column, centrifuging at 8000g for 1min, and removing the lower layer liquid; adding 500 μ L Buffer BW into the centrifugal column, centrifuging at 8000g for 1min, and removing the lower layer liquid; adding 500 μ L Buffer BD again, standing at room temperature for 15min, centrifuging at 8000g for 1min, and discarding the lower layer liquid; adding 500 μ L Buffer BW, centrifuging at 8000g for 1min, discarding the lower layer liquid, and repeating once; adding 250 μ L of 95% ethanol, centrifuging at 8000g for 1min, and removing the lower layer liquid; then, the mixture is centrifuged at 12000g at a high speed for 1min to completely remove the residual ethanol; the column was transferred to a new sterile 1.5mL centrifuge tube, 30. mu.L Buffer EB was added to the column, 8000g was centrifuged for 1min, the liquid in the tube was the purified DNA, and stored at-20 ℃ for future use.

Step 5.2, gene specific amplification:

fgf11, fgfrs2, pdgfr alpha and egfg7 gene promoter regions (Upstream 2k) obtained by high-throughput sequencing have regions with differential methylation, corresponding gene sequence fragments are obtained by controlling a reference genome, CpG Island which may exist is predicted by using a CpG Island Search program, gene specific primers are designed at both ends of the CpG Island to amplify specific fragments, and the PCR system is as follows: purified DNA 0.5. mu.L, 10 XTaq Buffer 2. mu. L, dNTP 0.5.5. mu.L, Taq polymerase 0.5. mu. L, F-primer 0.5. mu. L, R-primer 0.5. mu.L, sterile H₂O15.5. mu.L, placed in the following PCR program: 95 ℃ for 10min, 40 cycles (94 ℃ for 30s, 55 ℃ for 30s, 72 ℃ for 40s), 72 ℃ for 10 min.

Step 5.3, tapping and recycling PCR products:

the gel cutting recovery of the target fragment of the PCR product was carried out according to the protocol of the DNA purification recovery kit (Shanghai Czeri Co.), and the concentration thereof was measured by using a NanoDrop-1000 ultramicro UV spectrophotometer.

And 5.4, connecting with a carrier:

mu.L of the target fragment purified by tapping was added to a 10. mu.L system according to the instructions of the pMD19-T vector Ligation kit (TaKaRa), Ligation Solution I4.5. mu. L, PMD19-T vector 0.5 and sterile water 3. mu.L were added and ligated overnight at 16 ℃.

And 5.5, converting the target fragment:

transferring the connected product to a centrifuge tube filled with 100 mu L of DH5 alpha competent cells, and immediately placing on ice for 30min after flicking for a few times; then putting the mixture into a water bath kettle at 42 ℃ for heat shock treatment for 90s, immediately putting the mixture back on ice for cooling for 2min, adding 890 mu L of LB culture medium, and putting the mixture into a shaker at 37 ℃ for 200r/h to culture activated cells for 1 h. Sucking 100 μ L of the activated bacterial liquid, uniformly spreading on LB solid medium added with ampicillin, and placing the culture plate into a 37 ℃ incubator for inverted culture for 15 h.

Step 5.6, target fragment detection, inoculation culture and sequencing:

randomly picking 10 single colonies on the culture plate, and detecting whether the target fragment sequence exists by using a gene specific primer and an M13 primer. And (3) putting the colony with positive PCR detection into an LB liquid culture medium containing 1 per mill for amplification culture, and sending the colony to Shanghai Czeri company for sequencing to verify the methylation level of the single gene in different phenotypic characters.

Analysis of the results of example 1:

the methylation level refers to the ratio of the methylation number of three types (CG, CHG and CHH) covering C bases in the genome to the total methylated C bases, and the numerical value can be expressed as:

methylation level ═ (number of sequences covered to mC/total number of valid covered sequences) × 100%.

1. Genome-wide mean methylation level

The genome-wide mean methylation levels reflect the overall characteristics of the methylation profile of a particular species' genome, and the results are shown in Table 3.

TABLE 3

Sample (I)	C(％)	CG(％)	CHG(％)	CHH(％)
					L group	7.03	71.89	0.71	0.73
S group	7.04	72.01	0.68	0.7

It can be seen that the average methylation level of the whole genome of the species of the large yellow croaker is mainly CG type.

2. Genome-wide methylation horizontal distribution trend

2.1 distribution ratio of CG, CHG and CHH in methylated C base

At the genome-wide level, there is a large difference in the composition ratio of the three types of methylated cytosines (mCG, mCHG and mCHH) in different species. In particular, the number of each type of methylated C base and the proportion of all mC sites are important features of the whole genome methylation map of the species, and the results are shown in Table 4.

TABLE 4

	Total mCs	mCG	mCHG	mCHH
					Number of L groups	12769211	11900847	220327	648037
L population ratio (%)	100	93.2	1.73	5.07
					Number of S groups	12326383	11415822	231815	678746
S population ratio (%)	100	92.61	1.88	5.51

It can be seen that the highest proportion of CG-type methylation indicates that genomic methylation of the large yellow croaker population occurs mainly at CG sites.

2.2 methylation level distribution in methylated CG, CHG and CHH

The methylation level distribution of three types (CG, CHG and CHH) of C base in different species is different, so that the methylation level distribution of each type (mCG, mCHG and mCHH) of methylation is counted, and the DNA methylation characteristics of the species can be reflected. The results show that mCG sites are generally in a high methylation level state in the L population and the S population, namely most CG methyl sites are in a methylation level state of 90% -100%, and the CHG and CHH sites have relatively slow change trend, and most of the change trend is between 20% -50%.

2.3 methylation distribution characterization of different genomic regions

The methylation levels of the different genomic regions (Genebody, Upstream2k, Downstream2k, Exon, Intron, CDS, 5 'UTR, 3' UTR) have different respective biological functions in the genome. The results of the study show that the CG/CHG/CHH three types of the large yellow croaker genome are methylated most in the main gene region, are introns, and are methylated least in the 3' UTR.

2.4 DNA methylation levels in different transcription elements of the genome

All encoding gene sequences are divided into 3 different transcription element regions of upstream2k, genebody, and downstream2k, and the methylation level of the 3 different transcription element regions is counted. The results are shown in FIGS. 1-2.

3 differential methylation region analysis

3.1 DMRs related differential Gene statistics

And comparing the genes related to the DMRs obtained from the whole genome methylation sequencing information counted by the S population and the L population to obtain 2204 regions related differential methylation genes of the three types of DMRs of CG/CHG/CHH, wherein the up-regulated genes are 1506, and the down-regulated genes are 698. 2171 genes differentially methylated in CG/CHG/CHH three types, 1505 genes are up-regulated and 666 genes are down-regulated; the differentially methylated genes in the regions of the genes Upestream 2k, genebody and Downstem 2k are 333, 1609 and 229 respectively; 33 CHG type differential methylation genes, wherein 1 gene is up-regulated and 32 genes are down-regulated; the differentially methylated genes located in the regions of the genes Upstream2k, genebody and Downstem 2k were 1, 31 and 1, respectively. DMRs of CHH type were not screened for differentially methylation-associated genes.

3.2 GO enrichment analysis of genes related to DMRs

And respectively mapping the CG/CHG type DMRs to each sub-node of the GO database, calculating the gene number of each node, thereby obtaining a DMRs list of a certain GO subject obviously enriched by each differential marker gene and the number statistics thereof, and describing and analyzing according to three subjects of a participated Biological Process (BP), a Cell Component (CC) and a Molecular Function (MF).

3.2.1 GO enrichment analysis of CG-type DMRs associated genes

The Upstream2k region has 1899 CG-type DMRs (covering 333 differentially expressed genes) which are mapped to GO functional sub-nodes (related genes of the same DMRs can be mapped to a plurality of physiological processes on the GO sub-nodes, the same applies below), wherein 970 DMRs related genes which are accumulatively mapped to the category BP comprise 4 DMRs related genes which participate in the growth process and 86 DMRs related genes which participate in the development process; 649 DMRs-related genes mapped to CC category were accumulated, and the number of DMRs-related genes involved in cells and cell parts was 135 at most; 280 genes related to DMRs mapped to MF class were accumulated, 138 genes related to DMRs involved in the binding process and 1 gene related to DMRs involved in transcription factor activity-protein binding.

The Genebody region maps to 10335 CG-type DMRs on the GO functional subunit node (1609 differentially expressed genes are covered), wherein 5165 DMRs related genes of the BP category comprise 45 and 444 DMRs related genes involved in growth and development processes; the number of DMRs-associated genes in CC category was 3626, and the number of DMRs-associated genes involved in cells and cell parts was 747 and 746, respectively; there are 1208 genes related to DMRs in MF class, a maximum of 791 genes related to DMRs involved in binding process, and 1 gene related to DMRs in electron vector activity.

The Down stream2k region has 1317 CG-type DMRs (covering 229 differentially expressed genes) mapped to GO functional sub-nodes, wherein the number of genes related to the DMRs in the BP category is the largest, the number of genes related to the DMRs in the CC category is 641, the number of genes related to the DMRs in the MF category is 481, and the number of genes related to the DMRs in the MF category is 195. The number of DMRs-associated genes involved in growth and development in the BP class was 5 and 46, respectively.

3.2.2 GO enrichment analysis of CHG-type DMRs associated genes

In the Upstream2k region, only 1 gene (basic G-protein coupled receptor 19) related to the DMRs CHG type DMRs are mapped to the sub-nodes of BP, CC and MF of GO function, such as biological regulation, membrane and signal sensor activity.

A total of 177 DMRs of CHG type (covering 31 differentially expressed genes) mapped to sub-nodes of GO function in the Genebody region, with 83 differentially methylation-related genes for DMRs of the BP class, and 6 of them involved in the development process; there were 64 methylation-associated genes DMRs mapped to the CC class and 30 methylation-associated genes DMRs mapped to the MF class.

CHG type DMRs of 1 differentially methylated gene in the Down stream2k region are mapped to BP, CC and MF categories on a GO enrichment map, and have biological regulation, combination, organ parts, membranes, organelles, cells, cell parts and other sub-nodes respectively.

3.3 pathway enrichment analysis of genes related to DMRs

The pathway obviously enriched by the related Genes of the different DMRs is found by using a pathway public database of KEGG (Kyoto Encyclopedia of Genes and genomics, Kyoto Encyclopedia of Genes and Genomes) so as to determine the biochemical metabolism, signal transduction and other pathways involved by the related Genes of the DMRs.

3.3.1 pathway enrichment analysis of Gene CG-DMRs associated with the Upstream2k region

As shown in Table 5, CG-type DMRs in the region of the differentially methylation related gene upstream2k of the two populations S and L (S vs L) were annotated to the KEGG metabolic pathway, and there were 79 KEGG pathways and 175 DMRs annotated to the six major metabolic pathways of KEGG. The number of pathways involved in metabolism is at most (27), and there are 38 DMRs; the number of DMRs involved in cytological processes is maximum (44), and the pathway has 12 paths; the human disease pathway involves the least number of pathways and DMRs, 3 and 9 respectively.

TABLE 5

5 significant enrichment metabolic pathways with the P less than 0.05 are screened from the 79 KEGG pathways, and mainly participate in the processes of endocytosis, cytokine-cytokine receptor interaction, actin cytoskeleton regulation, N-glycan biosynthesis, focal adhesion and the like. Wherein the differential methylation enriched in endocytosis pathway reaches a very significant level (P < 0.01); the differential methylation of the regulatory pathway of actin cytoskeleton reaches a significant level (P < 0.05), and 8 differential methylation genes such as epidermal growth factor, EGFR, P21 activated kinase 1 and the like are screened in the pathway.

3.3.2 pathway enrichment analysis of Gene region-associated Gene CG-DMRs

As shown in Table 6, CG-type DMRs in the genebody region of the S and L two population differential methylation related genes are annotated into the KEGG pathway metabolic pathway, and 136 KEGG pathways and 1056 DMRs in the six metabolic pathways are annotated into the KEGG. The number of pathways involved in the metabolic pathway is at most (68 in total), and 226 DMRs are involved; the number of DMRs participating in the processing of the environment information is maximum (295), and the pathway comprises 17 paths; the human disease pathway involves the least number of pathways and DMRs, 4 and 65, respectively.

TABLE 6

KEGG main passage	KEGG subclass pathway gene number.	Number of differential unigenes
			Cytological procedures	14	245
Environmental information processing	17	295
			Genetic information processing	17	73
Human diseases	4	65
			Metabolism of matter	68	226
Organic system	16	152

17 significant enrichment metabolic pathways with the P less than 0.05 are screened from 1056 KEGG pathways with differential methylation enrichment, and mainly participate in pathways such as Notch signal pathways, focal adhesion, adhesion connection, MAPK signal pathways, TGF-beta signal pathways and the like. Wherein the P value and the Q value of 3 pathways enriched in Notch signal pathways, focal adhesion and adhesion connection reach extremely significant levels at the same time, and the P values of 7 signal pathways such as AGEs-RAGE signal pathways, insulin resistance and the like in ErbB signal pathways and diabetic complications reach extremely significant levels. Among them, DMRs of 41 growth-related genes such as fibroblast growth factor, fibroblast growth factor receptor, growth factor receptor binding protein, etc. are found in MAPK signaling pathway.

3.3.3 pathway enrichment analysis of CG-DMRs related to the region of Downstream2k Gene

As shown in Table 7, CG-type DMRs in the region of the two population (S vs L) differentially methylated gene downstream2k were annotated into the KEGG pathway metabolic pathway, resulting in 77 KEGG pathways and 162 DMRs annotated into the six major pathways of KEGG. Wherein the substance metabolism pathway involves 24 pathways at most, and 30 DMRs are total; the maximum number of DMRs (38) participating in the environmental information processing is 13 paths; the number of pathways involved in human disease is 4 and 14, which is the least of six metabolic pathways.

TABLE 7

KEGG main passage	KEGG subclass pathway gene number	Number of differential unigenes
			Cytological procedures	12	29
Environmental information processing	13	38
			Genetic information processing	10	20
Human diseases	4	14
			Metabolism of matter	24	30
Organic system	14	31

Of the 77 KEGG pathways, 5 significant enrichment metabolic pathways with P < 0.05 are concerned, and mainly participate in Notch signal pathways, Jak-STAT signal pathways, protein processing in endoplasmic reticulum, adipocyte factor signal pathways, herpes simplex infection and other pathways. Among them, DMRs of 5 genes such as Notch, DVL, CLS and the like were obtained by screening Notch signaling pathway species.

3.4 screening and quantitative PCR analysis of genes related to CG-DMRs growth

3.4.1 screening of genes related to CG-DMRs growth

13 genes related to growth traits were preliminarily screened on CG-type DMRs, shown in Table 8, for verification and analysis of quantitative PCR verification of gene mRNA differential expression level and whole genome BS sequencing results.

TABLE 8

3.4.2 analysis of relationship between results of qRT-PCR analysis and methylation level

The genes in Table 8 were analyzed for the relationship between the relative expression level of mRNA and its DNA methylation by qRT-PCR, and the results showed that the DNA methylation level in the genebody region was positively correlated with the relative expression level of gene mRNA, and the DNA methylation level in the uppstream 2k region was negatively correlated with the relative expression level of gene mRNA. The result shows that the sequencing result data of the whole genome BS is reliable, and the relationship between the methylation level of the growth related gene and the gene expression level can be accurately detected.

3.5 correlation analysis of Single Gene methylation level with growth traits

3.5.1 differential methylation Gene Screen

The upstream2k region was screened for 4 genes, fibroblast growth factor 11-like gene (fgf11), fibroblast growth factor receptor substrate 2(fibroblast growth factor receptor substrate 2, fgfrs2), epidermal growth factor-like protein 7(epidermal growth factor-like protein 7, egflp7), platelet-derived growth factor receptor alpha (pdgfr alpha).

3.5.2 differential methylated Gene amplification

Transforming sample muscle DNA by sulfite, amplifying by gene-specific BSP primer (shown as SEQ ID No5-SEQ ID No 12) to obtain an electropherogram, wherein the detection results of 65 samples of the fgfrs2 gene and fgf11 gene are shown in FIGS. 7-8, because the No. 65 sample is taken as the pre-amplification detection BSP primer for single electrophoresis, and BSP sequencing of 65 samples is carried out completely after the BSP primer is detected to be qualified, the electropherogram only contains the first 64 samples; the results of 20 samples of pdgfr alpha gene and egflp7 are shown in FIGS. 9-10.

3.5.3 Gene methylation region sequence information

The differential methylation region sequences of the 4 genes (fgf11, fgfrs2, egflp7 and pdgfr α) were obtained by gene cloning and sequencing and the results are shown in Table 1SEQ ID No.1-SEQ ID No. 4.

3.5.3 methylation sequencing results of single genes

fgf11 genes were significantly more methylated in the pedigree S population than in the pedigree L population, and fgf11 genes had an average methylation frequency of 0.5333 in the pedigree S population, significantly higher than in the pedigree L population (0.448), consistent with the genome-wide BS sequencing conclusions with reference to FIG. 3.

The methylation level of the fgfrs2 gene was significantly higher in the pedigree S population than in the pedigree L population, and the mean methylation frequency of the fgfrs2 gene was 0.5367 in the pedigree S population and significantly higher than in the pedigree L population (0.4383), consistent with the genome-wide BS sequencing conclusion with reference to fig. 4.

The level of methylation of the egflp7 gene was significantly higher in the pedigree S population than in the pedigree L population, and the mean methylation frequency of the egflp7 gene was 0.2532 in the pedigree S population, significantly higher than in the pedigree L population (0.1464), consistent with the genome-wide BS sequencing conclusion with reference to fig. 5.

The methylation level of the pdgfr α gene was significantly higher in the pedigree S population than in the pedigree L population, and the mean methylation frequency of the pdgfr α gene was 04033 in the pedigree S population, significantly higher than in the pedigree L population (0.2786), consistent with the genome-wide BS sequencing conclusion with reference to fig. 6.

Example 2:

the application of the molecular marker of DNA methylation of the large yellow croaker in the breeding of the large yellow croaker comprises the following steps:

step a, extracting DNA (DNA extraction kit of Shanghai Czeri company) from muscle tissues of large yellow croakers, converting the DNA with sulfite, and performing PCR amplification by using primers shown in (SEQ ID No.5-SEQ ID No.12) in the table 1;

and b, detecting the PCR amplification product, referring to the molecular marker fgf11, the molecular marker fgfrs2, the molecular marker egflp7 and the molecular marker pdgfr alpha shown in the table 1 (SEQ ID No.1-SEQ ID No.4), and selecting an individual with low average methylation frequency of fgf11 gene, fgfrs2 gene, egflp7 gene and pdgfr alpha gene as a breeding parent.

In summary, the method for screening molecular markers of DNA methylation of large yellow croaker provided by the present invention utilizes WGBS to detect DNA methylation characteristics of large yellow croaker, obtains genes related to differential methylation regions by screening methylation patterns of L and S populations of large yellow croaker, screens 13 growth related genes from CG-type DMRs related genes for analysis of the relationship between qRT-PCR and methylation frequency thereof, further screens 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 alpha (pdgfra) from 7 genes related to promoter region DMRs, wherein the methylation frequency of the 4 promoter growth factor genes in S population is higher than that in L population, and is in negative relationship with mRNA expression level of the genes, and methylation of promoter regions of these genes can directly regulate gene expression, the 4 genes are proved to have important regulation function on the cell growth, proliferation and individual growth and development of the large yellow croaker;

the molecular marker obtained by screening is applied to the production practice to assist breeding of the large yellow croaker, and provides important reference for molecular marker-assisted breeding and epigenetic regulation mechanism of the large yellow croaker;

the large yellow croaker DNA methylation molecular marker provided by the invention can be applied to production practice for auxiliary breeding research; the method can be applied to breeding of the large yellow croaker, is used for screening parent large yellow croaker, and can effectively improve the breeding speed of excellent large yellow croaker varieties, improve the germplasm of the large yellow croaker and improve the quality of fry.

The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all equivalent changes made by using the contents of the present specification and the drawings, or applied directly or indirectly to the related technical fields, are included in the scope of the present invention.

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Claims

1. The molecular marker for DNA methylation of the large yellow croaker is characterized by comprising a molecular marker fgf11, a molecular marker fgfrs2, a molecular marker egflp7 and a molecular marker pdgfr alpha;

the nucleotide sequence of the molecular marker fgf11 is shown as SEQ ID No. 1;

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

the nucleotide sequence of the molecular marker egflp7 is shown as SEQ ID No. 3;

the nucleotide sequence of the molecular marker pdgfr alpha is shown as SEQ ID No. 4.

2. The primer for detecting the molecular marker for DNA methylation of the large yellow croaker, according to claim 1, is characterized by comprising:

fgf11 upstream primer with nucleotide sequence shown as SEQ ID No. 5;

fgf11 downstream primer with nucleotide sequence shown as SEQ ID No. 6;

an upstream primer of fgfrs2, the nucleotide sequence of which is shown as SEQ ID No. 7;

a downstream primer of fgfrs2 with the nucleotide sequence shown as SEQ ID No. 8;

an upstream primer of egflp7 with the nucleotide sequence shown as SEQ ID No. 9;

an egflp7 downstream primer with the nucleotide sequence shown as SEQ ID No. 10;

a pdgfr α forward primer having the nucleotide sequence shown in SEQ ID No. 11;

the pdgfr alpha downstream primer has the nucleotide sequence shown in SEQ ID No. 12.

3. The method for screening the large yellow croaker DNA methylated molecular marker of claim 1, comprising the steps of:

and 4, verifying the single-gene DNA methylation level and growth character correlation of the differential methylation functional gene by adopting the primer disclosed by claim 2, obtaining DNA methylation sites with obvious regulation and control effects on the expression level of the growth functional gene, and obtaining the molecular marker of the DNA methylation of the large yellow croaker.

4. The method of claim 3, wherein the differentially methylated region in step 1 is a differentially methylated region of a methylated C site, and the differentially methylated region of a methylated C site has the following characteristics:

more than 5 methylated C bases are present in the region in at least one sample;

the requirement of the total sequencing depth of each methylated C site is more than 10, and the supported sequencing depth of each methylated C site is more than 4;

the length of the region should be between 40bp and 10 kb;

the distance between two adjacent methylation C sites is not more than 200 bp;

there is an average methylation level of more than 2 times;

the chi-square test P is less than or equal to 0.05.

5. The method for screening the molecular marker for DNA methylation of large yellow croaker according to claim 1, wherein the qRT-PCR primers in step 3 comprise:

fgf11 upstream primer with nucleotide sequence shown as SEQ ID No. 13;

fgf11 downstream primer with nucleotide sequence shown as SEQ ID No. 14;

an upstream primer of fgfrs2, the nucleotide sequence of which is shown as SEQ ID No. 15;

a downstream primer of fgfrs2 with the nucleotide sequence shown as SEQ ID No. 16;

an upstream primer of egflp7 with the nucleotide sequence shown as SEQ ID No. 17;

an egflp7 downstream primer with the nucleotide sequence shown as SEQ ID No. 18;

a pdgfr alpha upstream primer having the nucleotide sequence shown in SEQ ID No. 19;

the pdgfr alpha downstream primer has the nucleotide sequence shown in SEQ ID No. 20.

6. The use of the large yellow croaker DNA methylated molecular marker of claim 1 in large yellow croaker breeding, comprising the steps of:

step a, extracting DNA from muscle tissues of large yellow croaker, and carrying out PCR amplification by using the primer of claim 2 after sulfite conversion;

and b, detecting PCR amplification products, and selecting an individual with low average methylation frequency of fgf11 gene, fgfrs2 gene, egflp7 gene and pdgfr alpha gene as a breeding parent by referring to the molecular marker fgf11, the molecular marker fgfrs2, the molecular marker egflp7 and the molecular marker pdgfr alpha of claim 1.

7. The use of the molecular marker for DNA methylation of large yellow croaker according to claim 6, wherein the reaction system for sulfite conversion in step a is: mu.g DNA, 85. mu.L Bisulfite solution, 35. mu.L DNA protect buffer, RNase-free water was added to a volume of 140. mu.L.

8. The application of the large yellow croaker DNA methylated molecular marker of claim 6 in large yellow croaker breeding, wherein the PCR amplification conditions in step a are as follows: 10min at 95 ℃; 30s at 94 ℃, 30s at 55 ℃, 40s at 72 ℃ and 40 cycles; 10min at 72 ℃.