Application of methylation molecular marker combination related to chicken growth in breeding of high-adult-weight chicken

Abstract

The invention belongs to the technical field of gene engineering, particularly discloses application of a methylation molecular marker combination related to chicken growth in breeding of high-adult-weight chickens, and discovers a group of specific methylation gene molecular markers closely related to chicken weight characteristics and sequence primers for amplifying the molecular markers. The development of the molecular markers and the primers provides an effective molecular marker for the protection and early breeding of excellent characters of chickens, is beneficial to deeply analyzing the growth characters of the chickens from the new perspective of gene methylation modification, and promotes the progress of genetic improvement work of poultry.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, in particular to the application of a methylation molecular marker combination associated with chicken growth in breeding high adult weight chickens. Background Art

[0002] DNA methylation is an epigenetic modification that involves the addition of methyl groups to cytosine residues in DNA molecules, usually on CpG islands. This modification is crucial for biological processes such as gene expression regulation, cell differentiation, developmental processes, and X chromosome inactivation. DNA methylation is mainly catalyzed by DNA methyltransferases (DNMTs), which use S-adenosylmethionine (SAM) as a methyl donor to transfer the methyl group to the 5' position of cytosine to form 5'-methylcytosine. Methylation plays a central role in regulating gene expression, usually inhibiting gene transcription, thereby affecting gene expression regulation. Due to its high stability, DNA methylation can be considered as part of the genetic system and may affect the evolutionary characteristics of mammals induced by environmental factors. Studies have shown that DNA methylation is a key regulatory mechanism for genomic adaptability and phenotypic variation in response to environmental stimuli and artificial selection. The transgenerational transmission of methylation variation often occurs in highly selected regions or sites, and general selected sites or random mutation sites will not be transmitted. The study also found that some of the epigenetic modifications produced by selection are independent of DNA variation, and they can play an independent role in the process of artificial domestication and growth performance improvement. This shows that when environmental stimulation and artificial selection occur, the intergenerational transmission of epigenetic modifications and their synergistic effect with genomic variation are important mechanisms for adaptive changes in animal traits.

[0003] Traditional whole genome methylation sequencing (WGBS) technology is expensive and not convenient for large-scale methylation analysis. Although reduced representational bisulfite sequencing (RRBS) can reduce costs, it mainly targets CpG-rich regions and cannot fully cover the methylation information in the genome. In the process of chicken germplasm resource protection and excellent breed selection, predecessors have conducted a certain degree of research on the mutation sites of candidate genes and their effects on chicken growth characteristics. Despite this, there is still a lack of in-depth understanding of the multi-gene interaction mechanism involved in the improvement of chicken growth traits and how DNA methylation and genomic variation jointly regulate growth traits. Therefore, the development of methylation gene markers related to chicken growth is of great significance for guiding the selection and breeding of excellent chicken breeds. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides the application of a combination of methylation molecular markers associated with chicken growth in breeding high adult weight chickens.

[0005] To achieve the above object, the present invention is implemented according to the following technical solutions:

[0006] One of the technical solutions of the present invention is an application of a methylation molecular marker combination related to chicken growth in breeding chicken varieties with high weight and growth rate, wherein the methylation molecular marker combination comprises:

[0007] The molecular marker DMR5 located at position 394713-394803 of the nucleotide sequence of the chicken chromosome chr01;

[0008] The molecular marker DMR7.1 located at position 419621-419805 of the nucleotide sequence of the chicken chromosome chr01;

[0009] The molecular marker DMR3 located at position 51975700-51975847 of the nucleotide sequence of the chicken chromosome chr01;

[0010] The molecular marker DMR1.2 located at position 128238988-128239164 of the nucleotide sequence of the chicken chromosome chr01;

[0011] The molecular marker DMR2 located at position 130235456-130235580 of the nucleotide sequence of the chicken chromosome chr01;

[0012] The molecular marker DMR78.2 located at position 168993706-168993810 of the nucleotide sequence of chicken chromosome chr01;

[0013] The molecular marker DMR92 located at position 169025095-169025265 of the nucleotide sequence of the chicken chromosome chr01;

[0014] The molecular marker DMR95.1 located at the nucleotide sequence of chicken chromosome chr01 at position 169084779-169084966;

[0015] The molecular marker DMR79.2 located at position 169188293-169188469 of the nucleotide sequence of chicken chromosome chr01;

[0016] The molecular marker DMR79.4 located at position 169188645-169188821 of the nucleotide sequence of the chicken chromosome chr01;

[0017] The molecular marker DMR79.5 located at position 169188821-169189001 of the nucleotide sequence of the chicken chromosome chr01;

[0018] The molecular marker DMR9.5 located at the nucleotide sequence of chicken chromosome chr01 at position 175916030-175916200;

[0019] The molecular marker DMR12 located at position 175918197-175918348 of the nucleotide sequence of chicken chromosome chr01;

[0020] The molecular marker DMR14 located at position 37653446-37653644 of the nucleotide sequence of chicken chromosome chr02;

[0021] The molecular marker DMR13 located at position 62705794-62705922 of the nucleotide sequence of chicken chromosome chr02;

[0022] Molecular marker DMR15 located at positions 86234800-86234838 of the nucleotide sequence of chicken chr02 chromosome; molecular marker DMR16.1 located at positions 39683111-39683246 of the nucleotide sequence of chicken chr03 chromosome; molecular marker DMR16.2 located at positions 39683246-39683381 of the nucleotide sequence of chicken chr03 chromosome; molecular marker DMR17 located at positions 54606636-54606703 of the nucleotide sequence of chicken chr03 chromosome; molecular marker DMR18.3 located at positions 43933331-43933479 of the nucleotide sequence of chicken chr04 chromosome ; Molecular marker DMR20 located at positions 49073674-49073756 of the chicken chr04 chromosome nucleotide sequence; molecular marker DMR25.1 located at positions 64949115-64949248 of the chicken chr04 chromosome nucleotide sequence; molecular marker DMR25.3 located at positions 64949381-64949516 of the chicken chr04 chromosome nucleotide sequence; molecular marker DMR21 located at positions 65963314-65963394 of the chicken chr04 chromosome nucleotide sequence; molecular marker DMR77 located at positions 75708478-75708558 of the chicken chr04 chromosome nucleotide sequence; The molecular marker DMR89 is located at the 75722058-75722181 position of the nucleotide sequence of the chicken chr04 chromosome; the molecular marker DMR90.1 is located at the 75865368-75865535 position of the nucleotide sequence of the chicken chr04 chromosome; the molecular marker DMR91.2 is located at the 75883037-75883221 position of the nucleotide sequence of the chicken chr04 chromosome; the molecular marker DMR28.3 is located at the 35706050-35706173 position of the nucleotide sequence of the chicken chr05 chromosome; the molecular marker DMR35 is located at the 11841291-11841407 position of the nucleotide sequence of the chicken chr07 chromosome .1; molecular marker DMR36.4 located at positions 12347383-12347547 of the nucleotide sequence of chicken chromosome chr07; molecular marker DMR29 located at positions 21698914-21699104 of the nucleotide sequence of chicken chromosome chr07; molecular marker DMR30 located at positions 21699220-21699403 of the nucleotide sequence of chicken chromosome chr07; molecular marker DMR39 located at positions 22031214-22031341 of the nucleotide sequence of chicken chromosome chr07; molecular marker DMR32 located at positions 27979214-27979401 of the nucleotide sequence of chicken chromosome chr07.3; molecular marker DMR32.4 located at the 27979401-27979588 position of the nucleotide sequence of chicken chr07 chromosome; molecular marker DMR44.1 located at the 24790785-24790940 position of the nucleotide sequence of chicken chr08 chromosome; molecular marker DMR45.3 located at the 4788438-4788615 position of the nucleotide sequence of chicken chr09 chromosome; molecular marker DMR45.4 located at the 145 The molecular marker DMR46.2 located at positions 93243-14593394; the molecular marker DMR49.1 located at positions 5068455-5068653 of the nucleotide sequence of the chicken chr10 chromosome; the molecular marker DMR50.4 located at positions 1265653-1265851 of the nucleotide sequence of the chicken chr11 chromosome; the molecular marker DMR52 located at positions 1332269-1332420 of the nucleotide sequence of the chicken chr11 chromosome;.

[0023] The molecular marker DMR53 located at the nucleotide sequence 1332609-1332668 of the chicken chromosome chr11;

[0024] The molecular marker DMR55 located at the nucleotide sequence 9956394-9956428 of the chicken chromosome chr13;

[0025] The molecular marker DMR75 is located at the 17387181-17387313 position of the chicken chr13 chromosome nucleotide sequence; the molecular marker DMR76 is located at the 17393714-17393806 position of the chicken chr13 chromosome nucleotide sequence; the molecular marker DMR88.1 is located at the 17397928-17398116 position of the chicken chr13 chromosome nucleotide sequence; the molecular marker DMR88.2 is located at the 17398116-17398304 position of the chicken chr13 chromosome nucleotide sequence; the molecular marker DMR88.3 is located at the 17398304-17398494 position of the chicken chr13 chromosome nucleotide sequence; the molecular marker DMR57 is located at the 2010808-2010939 position of the chicken chr16 chromosome nucleotide sequence;

[0026] The molecular marker DMR56 located at the nucleotide sequence 2011191-2011350 of the chicken chromosome chr16;

[0027] The molecular marker DMR58 located at the nucleotide sequence 2045076-2045199 of the chicken chromosome chr17;

[0028] The molecular marker DMR59.2 located at the 6468876-6469072 position of the chicken chr17 chromosome nucleotide sequence; the molecular marker DMR84 located at the 10050540-10050564 position of the chicken chr17 chromosome nucleotide sequence; the molecular marker DMR60.4 located at the 6294431-6294587 position of the chicken chr21 chromosome nucleotide sequence; the molecular marker DMR82.1 located at the 3260665-3260791 position of the chicken chr27 chromosome nucleotide sequence; the molecular marker DMR83 located at the 3280720-3280825 position of the chicken chr27 chromosome nucleotide sequence;

[0029] The molecular marker DMR61 located at the nucleotide sequence 1013255-1013371 of the chicken chromosome chr34;

[0030] The molecular marker DMR62.1 located at position 287402-287511 of the nucleotide sequence of chicken chromosome chr35;

[0031] The molecular marker DMR63 located at the nucleotide sequence 1240367-1240514 of the chicken chrZ chromosome;

[0032] The molecular marker DMR73 located at the 14076891-14077028 position of the nucleotide sequence of the chicken chrZ chromosome; the molecular marker DMR69 located at the 14281710-14281765 position of the nucleotide sequence of the chicken chrZ chromosome;

[0033] The molecular marker DMR64 located at the nucleotide sequence 22509085-22509262 of the chicken chrZ chromosome;

[0034] The molecular marker DMR65.9 located at position 22513943-22514130 of the nucleotide sequence of the chicken chrZ chromosome;

[0035] The molecular marker DMR67.1 located at the nucleotide sequence 23240108-23240284 of the chicken chrZ chromosome;

[0036] The molecular marker DMR68.1 located at the nucleotide sequence 25575489-25575681 of the chicken chrZ chromosome;

[0037] The molecular marker DMR66.1 located at position 44659630-44659788 of the nucleotide sequence of the chicken chrZ chromosome;

[0038] The molecular marker DMR70.4 is located at position 57389921-57390087 of the nucleotide sequence of chicken chrZ chromosome.

[0039] The second technical solution of the present invention is a method for breeding high adult weight chickens using a combination of methylated molecular markers related to chicken growth, comprising the following steps:

[0040] S1. Extracting DNA from the blood of the individual chicken to be tested;

[0041] S2, methylating the extracted DNA to obtain a methylated product;

[0042] S3, designing specific primers according to the methylation molecular marker combination, using the methylation treatment product as a template, and performing multiple PCR amplification using specific amplification primers to obtain amplification products;

[0043] S4. The methylation level of the amplified product is tested, and then a score is calculated based on its methylation level. The scoring mechanism is established based on a set of thresholds for gene methylation levels that are closely related to body weight and growth rate. If the comprehensive score of an individual chicken exceeds this threshold, it will be selected as a breeding object.

[0044] Specifically, an upstream primer and a downstream primer are designed corresponding to each methylation molecular marker; the primers of all methylation molecular markers are shown in SEQ ID NO.1-SEQ ID NO.136 in sequence.

[0045] Specifically, the multiplex PCR reaction system: 8.5 μL of methylated product, 4 μL of methylated multiplex PCR primer mixture (primer dry powder was diluted and mixed at a concentration of 0.2 μmol each), 12.5 μL of 2×Multiplex Mix; reaction conditions were: 99°C for 2 min; 99°C for 15 s, 60°C for 4 min, 27 cycles; 72°C for 10 min; stored at 4°C; after the reaction, purification was performed using AgencourtAMPure XPBeads to obtain 10.5 μL of PCR reaction product.

[0046] Specifically, step S4 specifically includes: after sequencing the amplified product, filtering and aligning the sequencing data, and calculating the methylation level of each marker, constructing a Logistic regression model based on the Lasso method through the glmnet package in the R language, and calculating the accuracy of judging the individual as a high-weight breeding chicken based on the methylation degree of the above 68 molecular markers. If the accuracy of the rooster exceeds 76% and the accuracy of the hen exceeds 69%, the individual is judged as a high adult weight selection object.

[0047] Compared with the prior art, the present invention has discovered a group of specific methylation gene molecular markers that are closely related to chicken weight, as well as sequence primers for amplifying these molecular markers; the development of these molecular markers and primers provides an effective molecular marker for the protection and breeding of excellent traits of chickens, helps to deeply analyze the growth traits of chickens from a new perspective of methylation genes, and promotes the progress of poultry genetic improvement. DETAILED DESCRIPTION

[0048] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments. The specific embodiments described herein are only used to explain the present invention and are not used to limit the invention.

[0049] This example discovered a group of specific methylation gene molecular marker combinations that are closely related to the adult weight of Qingyuan Ma chickens. The methylation gene molecular markers are composed of 68 methylation regions on the genome, and the locations of the methylation regions on the genome are shown in Table 1.

[0050] Table 1

[0051]

[0052]

[0053]

[0054] The above-mentioned methylation molecular marker combination related to chicken growth is used to breed high adult weight chickens. The specific process is as follows:

[0055] S1. Blood samples of 20 males and 20 females were collected from 3 Qingyuan Ma chicken strains or generations with different degrees of selection for body weight at 120 days of age, totaling 120 individuals, and blood DNA was extracted;

[0056] S2, methylating the extracted DNA to obtain a methylated product;

[0057] An exemplary method of methylation treatment is:

[0058] a) Extract DNA from samples and prepare CT Conversion Reagent;

[0059] b) Take a PCR tube and add 200 ng DNA to 40 ul of water, add 110 μL CT Conversion Reagent and mix well, and centrifuge briefly to ensure that there is no residual liquid on the tube cap and tube wall;

[0060] c) Place the above PCR tube on a PCR instrument and run the following program: heat cover at 105°C; 98°C for 10 min; 64°C for 150 min (2.5 h); store at 4°C;

[0061] d) Place the No. 1 C purification column in the collection tube and add 600 μL of binding solution to the No. 1 C purification column.

[0062] e) Add all the reaction solution in step c directly to the purification column No. 1C and mix with the binding solution, cover the column and invert to mix. Centrifuge at ≥10,000×g for 1 minute and discard the filtrate;

[0063] f) Add 100 μL of the prepared washing solution to the No. 1 C purification column and centrifuge at ≥10,000r for 1 minute;

[0064] g) Add 200 μL of desulfurization solution to No. 1 C purification column, place at room temperature (20-30°C) for 15-20 minutes, and centrifuge at ≥10,000×g for 1 minute;

[0065] h) Add 200 μL of the prepared washing solution to the No. 1 C purification column and centrifuge at ≥10,000r for 1 minute;

[0066] i) repeat step h;

[0067] j) Transfer the No. 1 C purification column to a clean 1.5 ml centrifuge tube and pipette 10 μL of the eluent directly into the column matrix. Centrifuge at full speed for 1 minute to elute the DNA.

[0068] S3. Design specific primers according to the methylation molecular marker combination, as shown in Table 2;

[0069] Table 2

[0070]

[0071]

[0072]

[0073]

[0074] Using the methylated product as a template, multiple PCR amplification was performed using specific amplification primers to obtain amplified products; the multiple PCR reaction system was configured: 8.5 μL of methylated product, 4 μL of multiple PCR primer mixture (primer dry powder was diluted and mixed at a concentration of 0.2 μmol per primer), and 12.5 μL of 2×Multiplex Mix. The reaction conditions were: 99°C for 2 min; 99°C for 15 s, 60°C for 4 min, 27 cycles; 72°C for 10 min; stored at 4°C; after the reaction, it was purified using AgencourtAMPure XP Beads to obtain 10.5 μL of PCR reaction product.

[0075] S4. Sequencing library was constructed for multiplex PCR reaction products. The library construction reaction system was configured as follows: 10.5 μL of multiplex PCR reaction products, 2 μL of universal primers P5 and P7 (10 pmol), and 12.5 μL of 2×Multiplex Mix. The reaction conditions were: 98°C for 1 min; 98°C for 15 s, 60°C for 30 s, 72°C for 30 s, 12 cycles; 72°C for 1 min; and stored at 4°C. Among them, the sequences of universal primers P5 and P7 were as follows: P5: AATGATACGGCGACCACCGAGATCTACAC-INDEX: XXXXXXXX; P7: CAAGCAGAAGACGGCATACGAGAT-INDEX: XXXXXXXX; among them, INDEX is the tag sequence used to identify different specimens. After the reaction, AgencourtAMPure XP Beads was used for purification to obtain 30 μL of library product. Library sequencing can choose high-throughput sequencers of different models on platforms such as Illumina and BGI. After the sequencing is completed, the sequencing data is filtered, aligned, and the methylation level of each gene marker is calculated. The final effective result is not less than 30× depth. After the sequencing is completed, the sequencing data is filtered, aligned, and the methylation level of each marker is calculated. The logistic regression model based on the Lasso method is constructed through the glmnet package in the R language to calculate the accuracy of judging the individual as a high-weight breeding chicken based on the methylation degree of the above 68 molecular markers. If the accuracy rate of the rooster exceeds 76% and the accuracy rate of the hen exceeds 69%, the individual is judged as a high adult weight selection object.

[0076] In this example, we compared the effects of joint detection of 68 methylation molecular markers and detection of each methylation molecular marker individually. Table 3 shows the methylation levels of the above markers in 3 high-weight breeding individuals and 3 low-weight breeding individuals.

[0077] Table 3

[0078]

[0079]

[0080]

[0081] The effects of combined detection and single detection of methylation molecular markers are shown in Table 4. It can be seen from Table 4 that the combined detection of these 68 molecular markers shows higher specificity and sensitivity, and its detection performance is significantly better than that of detecting any molecular marker alone.

[0082] Table 4

[0083]

[0084]

[0085] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. Application of a methylation molecular marker combination associated with chicken growth in breeding high adult weight chickens, characterized in that: The methylation molecular marker combination comprises: The molecular marker DMR5 located at position 394713-394803 of the nucleotide sequence of the chicken chromosome chr01; The molecular marker DMR7.1 located at position 419621-419805 of the nucleotide sequence of the chicken chromosome chr01; The molecular marker DMR3 located at position 51975700-51975847 of the nucleotide sequence of the chicken chromosome chr01; The molecular marker DMR1.2 located at position 128238988-128239164 of the nucleotide sequence of the chicken chromosome chr01; The molecular marker DMR2 located at position 130235456-130235580 of the nucleotide sequence of the chicken chromosome chr01; The molecular marker DMR78.2 located at position 168993706-168993810 of the nucleotide sequence of chicken chromosome chr01; The molecular marker DMR92 located at position 169025095-169025265 of the nucleotide sequence of the chicken chromosome chr01; The molecular marker DMR95.1 located at the nucleotide sequence of chicken chromosome chr01 at position 169084779-169084966; The molecular marker DMR79.2 located at position 169188293-169188469 of the nucleotide sequence of chicken chromosome chr01; The molecular marker DMR79.4 located at position 169188645-169188821 of the nucleotide sequence of the chicken chromosome chr01; The molecular marker DMR79.5 located at position 169188821-169189001 of the nucleotide sequence of the chicken chromosome chr01; The molecular marker DMR9.5 located at the nucleotide sequence of chicken chromosome chr01 at position 175916030-175916200; The molecular marker DMR12 located at position 175918197-175918348 of the nucleotide sequence of chicken chromosome chr01; The molecular marker DMR14 located at position 37653446-37653644 of the nucleotide sequence of chicken chromosome chr02; The molecular marker DMR13 located at position 62705794-62705922 of the nucleotide sequence of chicken chromosome chr02; The molecular marker DMR15 located at position 86234800-86234838 of the nucleotide sequence of chicken chromosome chr02; The molecular marker DMR16.1 located at position 39683111-39683246 of the nucleotide sequence of chicken chromosome chr03; The molecular marker DMR16.2 located at position 39683246-39683381 of the nucleotide sequence of chicken chromosome chr03; The molecular marker DMR17 located at position 54606636-54606703 of the nucleotide sequence of chicken chromosome chr03; The molecular marker DMR18.3 located at position 43933331-43933479 of the nucleotide sequence of chicken chromosome chr04; The molecular marker DMR20 located at position 49073674-49073756 of the nucleotide sequence of chicken chromosome chr04; The molecular marker DMR25.1 located at position 64949115-64949248 of the nucleotide sequence of chicken chromosome chr04; The molecular marker DMR25.3 located at position 64949381-64949516 of the nucleotide sequence of chicken chromosome chr04; The molecular marker DMR21 located at position 65963314-65963394 of the nucleotide sequence of chicken chromosome chr04; The molecular marker DMR77 located at position 75708478-75708558 of the nucleotide sequence of chicken chromosome chr04; The molecular marker DMR89 located at the nucleotide sequence 75722058-75722181 of the chicken chromosome chr04; The molecular marker DMR90.1 located at position 75865368-75865535 of the nucleotide sequence of chicken chromosome chr04; The molecular marker DMR91.2 located at position 75883037-75883221 of the nucleotide sequence of chicken chromosome chr04; The molecular marker DMR28.3 located at position 35706050-35706173 of the nucleotide sequence of chicken chromosome chr05; The molecular marker DMR35.1 located at position 11841291-11841407 of the nucleotide sequence of chicken chromosome chr07; The molecular marker DMR36.4 located at position 12347383-12347547 of the nucleotide sequence of chicken chromosome chr07; The molecular marker DMR29 located at position 21698914-21699104 of the nucleotide sequence of chicken chromosome chr07; The molecular marker DMR30 located at position 21699220-21699403 of the nucleotide sequence of chicken chromosome chr07; The molecular marker DMR39 located at position 22031214-22031341 of the nucleotide sequence of chicken chromosome chr07; The molecular marker DMR32.3 located at position 27979214-27979401 of the nucleotide sequence of chicken chromosome chr07; The molecular marker DMR32.4 located at position 27979401-27979588 of the nucleotide sequence of chicken chromosome chr07; The molecular marker DMR44.1 located at position 24790785-24790940 of the nucleotide sequence of chicken chromosome chr08; The molecular marker DMR45.3 located at position 4788438-4788615 of the nucleotide sequence of chicken chromosome chr09; The molecular marker DMR46.2 located at position 14593243-14593394 of the nucleotide sequence of chicken chromosome chr09; The molecular marker DMR49.1 located at the nucleotide sequence 5068455-5068653 of the chicken chromosome chr10; The molecular marker DMR50.4 located at position 1265653-1265851 of the nucleotide sequence of chicken chromosome chr11; The molecular marker DMR52 located at the nucleotide sequence 1332269-1332420 of the chicken chromosome chr11; The molecular marker DMR53 located at the nucleotide sequence 1332609-1332668 of the chicken chromosome chr11; The molecular marker DMR55 located at the nucleotide sequence 9956394-9956428 of the chicken chromosome chr13; The molecular marker DMR75 located at the nucleotide sequence 17387181-17387313 of the chicken chromosome chr13; The molecular marker DMR76 located at the nucleotide sequence 17393714-17393806 of the chicken chromosome chr13; The molecular marker DMR88.1 located at the nucleotide sequence 17397928-17398116 of the chicken chromosome chr13; The molecular marker DMR88.2 located at the nucleotide sequence 17398116-17398304 of the chicken chromosome chr13; The molecular marker DMR88.3 located at position 17398304-17398494 of the nucleotide sequence of chicken chromosome chr13; The molecular marker DMR57 located at position 2010808-2010939 of the nucleotide sequence of chicken chromosome chr16; The molecular marker DMR56 located at the nucleotide sequence 2011191-2011350 of the chicken chromosome chr16; The molecular marker DMR58 located at the nucleotide sequence 2045076-2045199 of the chicken chromosome chr17; The molecular marker DMR59.2 located at the nucleotide sequence 6468876-6469072 of the chicken chromosome chr17; The molecular marker DMR84 located at position 10050540-10050564 of the nucleotide sequence of chicken chromosome chr17; The molecular marker DMR60.4 located at position 6294431-6294587 of the nucleotide sequence of chicken chromosome chr21; The molecular marker DMR82.1 located at position 3260665-3260791 of the nucleotide sequence of chicken chromosome chr27; The molecular marker DMR83 located at the nucleotide sequence 3280720-3280825 of the chicken chromosome chr27; The molecular marker DMR61 located at the nucleotide sequence 1013255-1013371 of the chicken chromosome chr34; The molecular marker DMR62.1 located at position 287402-287511 of the nucleotide sequence of chicken chromosome chr35; The molecular marker DMR63 located at the nucleotide sequence 1240367-1240514 of the chicken chrZ chromosome; The molecular marker DMR73 located at the nucleotide sequence 14076891-14077028 of the chicken chrZ chromosome; The molecular marker DMR69 located at the nucleotide sequence 14281710-14281765 of the chicken chrZ chromosome; The molecular marker DMR64 located at the nucleotide sequence 22509085-22509262 of the chicken chrZ chromosome; The molecular marker DMR65.9 located at position 22513943-22514130 of the nucleotide sequence of the chicken chrZ chromosome; The molecular marker DMR67.1 located at the nucleotide sequence 23240108-23240284 of the chicken chrZ chromosome; The molecular marker DMR68.1 located at position 25575489-25575681 of the nucleotide sequence of the chicken chrZ chromosome; The molecular marker DMR66.1 located at position 44659630-44659788 of the nucleotide sequence of the chicken chrZ chromosome; The molecular marker DMR70.4 is located at position 57389921-57390087 of the nucleotide sequence of chicken chrZ chromosome.

2. A method for breeding high adult weight chickens using a combination of methylated molecular markers associated with chicken growth, characterized in that: The following steps are involved: S1. Extracting DNA from the blood of the individual chicken to be tested; S2, methylating the extracted DNA to obtain a methylated product; S3, designing specific primers according to the methylation molecular marker combination, using the methylation treatment product as a template, and performing multiple PCR amplification using specific amplification primers to obtain amplification products; S4. The methylation level of the amplified product is tested, and then a score is calculated based on its methylation level. The scoring mechanism is established based on a set of thresholds for gene methylation levels related to adult body weight; if the comprehensive score of an individual chicken exceeds this threshold, it will be selected as a breeding object.

3. The method for breeding high adult weight chickens using a combination of methylated molecular markers associated with chicken growth according to claim 2, characterized in that: An upstream primer and a downstream primer are designed for each methylation molecular marker; the primers of all methylation molecular markers are shown in SEQ ID NO.1-SEQ ID NO.136 in sequence.

4. The method for breeding high adult weight chickens using a combination of methylated molecular markers associated with chicken growth according to claim 2, characterized in that: Multiplex PCR reaction system: 8.5 μL of methylated product, 4 μL of methylated multiplex PCR primer mixture, 12.5 μL of 2×Multiplex Mix; reaction conditions: 99°C for 2 min; 99°C for 15 s, 60°C for 4 min, 27 cycles; 72°C for 10 min; stored at 4°C; after the reaction, purification with AgencourtAMPure XP Beads can obtain 10.5 μL of PCR reaction product.

5. The method for breeding high adult weight chickens using a combination of methylated molecular markers associated with chicken growth according to claim 2, characterized in that: The step S4 specifically includes: after sequencing the amplified product, filtering and aligning the sequencing data, and calculating the methylation level of each marker, constructing a Logistic regression model based on the Lasso method through the glmnet package in the R language, and calculating the accuracy of judging the individual as a high-weight breeding chicken based on the methylation degree of the above 68 molecular markers. If the accuracy rate of the rooster exceeds 76% and the accuracy rate of the hen exceeds 69%, the individual is judged as a high adult weight selection object.

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