An egr1 gene molecular marker related to chicken carcass traits, a kit and application thereof

Abstract

The application discloses a kind of EGR1 gene molecular markers related to chicken carcass character, kit and its application, belong to biotechnology field.The nucleotide sequence of the molecular marker is as shown in SEQ ID NO:3, the 736th nucleotide sequence is SNP site, and G / C mutation exists;The SNP site exists CC, GC, GG three genotypes.Use the molecular marker to detect the genome DNA of chicken, and it is found through experiment that the SNP site rs313977419G> C of EGR1 gene sequence, the SNP site is significantly related to chicken carcass character.Therefore, the application can accurately identify chicken carcass character, and provide new molecular marker for molecular marker assisted selection breeding.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to an EGR1 gene molecular marker related to chicken carcass traits, a kit and application thereof. BACKGROUND

[0002] Single Nucleotide Polymorphism (SNP) refers to the polymorphism of genomic DNA sequence caused by the insertion, deletion, transversion and conversion of a single nucleotide at the genomic level. SNP is the most common type of heritable variation in animals, widely exists in animal genomes, and has the characteristics of stable inheritance and easy detection. In animal production practice, SNP can be used for molecular marker-assisted selection (MAS), thereby breaking through the bottleneck of traditional breeding to improve the accuracy of selection and the breeding effect of target traits.

[0003] EGR1(early growth response-1) gene is widely present in mammalian and human cells, belongs to the immediate early gene family, can be rapidly and transiently activated after a series of external stimuli, as a transcription factor, regulates the expression of many target genes by binding to specific sites on the target gene promoter, recent studies have shown that EGR1 may have an important impact on muscle growth and development. Zhang Weiwie et al. research shows that EGR1 can bind to the MYOG gene promoter region, promote the differentiation of bovine skeletal muscle satellite cells cultured in vitro by regulating the expression of MYOG gene. This suggests that EGR1 may be involved in the regulation of skeletal muscle development. Nie Jingru et al. research shows that the EGR1 gene is down-regulated in large Diqing Tibetan pigs during the growth stage S1 vs S2, inhibiting myogenic differentiation of myoblasts, reducing tendon collagen fibers and reducing muscle growth potential. Li Zixiong et al. research shows that injecting miR-499-5p can change the diameter of chicken gastrocnemius muscle fibers and thus affect muscle fiber characteristics, and the EGR1 gene in the differentially expressed genes may be involved in the regulation of fat deposition and muscle development under the action of miR-499-5p. Hong Guangliang found that EGR1 is involved in the development of skeletal muscle satellite cells and mainly promotes their differentiation in the study of the regulation of skeletal muscle satellite cell proliferation and differentiation. Then, the down-regulation of EGR1 expression is speculated to be conducive to inhibiting PSCs differentiation. Cheng Zimin found that EGR1 and FOS genes encoding transcription factors affect muscle growth and development by regulating cell proliferation, differentiation and apoptosis when exploring the regulatory mechanism of muscle fiber traits difference between Ma Sheng pig and Large White pig. Cui Yafeng et al. research found that Egr1 expression is located in the nucleus and cytoplasm of C2C12, and its expression in the nucleus and cytoplasm is significantly increased with differentiation, and after activation or inhibition of Egr1, the myotube fusion rate of C2C12 cells and the expression levels of myogenic markers myogenin and myosin heavy chain 2 are significantly increased or decreased, indicating that Egr1 can promote the differentiation of mouse myoblast C2C12 in vitro. It can be seen that EGR1 plays an important role in the process of muscle development, but there is no report on the correlation between EGR1 gene and chicken carcass traits. SUMMARY

[0004] The purpose of the present application is to provide an EGR1 gene molecular marker related to chicken carcass traits, a kit and its application, which solves the problems existing in the prior art, the molecular marker is located in the CDs region of EGR1 gene, and there is a SNP site related to chicken carcass traits, which provides a new SNP molecular marker for MAS.

[0005] To achieve the above purpose, the present application provides the following scheme:

[0006] The present application provides a molecular marker related to chicken carcass traits, the nucleotide sequence of the molecular marker is shown as SEQ ID NO: 3, and a SNP site exists at position 736 of the nucleotide sequence, and a G / C mutation exists.

[0007] Preferably, the SNP site has three genotypes of CC, GC and GG.

[0008] The present application also provides a primer pair for amplifying the molecular marker, and the nucleotide sequences of the primer pair are shown as SEQ ID NO: 1-2.

[0009] The present application also provides a kit for identifying chicken carcass traits, and the kit comprises the primer pair.

[0010] The present application also provides a method for identifying chicken carcass traits by using the molecular marker, which comprises the steps of amplifying the molecular marker or a gene sequence containing the molecular marker by PCR, analyzing the genotype of the SNP site of the molecular marker, and identifying the chicken carcass traits according to the genotype; the chicken carcass traits include live weight, half-eviscerated weight, whole-eviscerated weight, wing weight, leg weight and breast muscle rate.

[0011] Preferably, the reaction system of the PCR amplification is as follows: 2 μL of template DNA, 25 μL of amplification buffer, 2 μL of upstream primer, 2 μL of downstream primer and 19 μL of ddH2O.

[0012] Preferably, the reaction procedure of the PCR amplification is as follows: pre-denaturation at 94 ℃ for 2 min; denaturation at 94 ℃ for 30 s, annealing at 58 ℃ for 30 s, extension at 72 ℃ for 30 s, 32 cycles; final extension at 72 ℃ for 3 min; and preservation at 4 ℃.

[0013] Preferably, when the SNP site is of the GG genotype, the live weight, half-eviscerated weight, whole-eviscerated weight, wing weight and leg weight of the chicken are high; and when the SNP site is of the GC genotype, the breast muscle rate of the chicken is high.

[0014] The present application also provides application of the molecular marker, or the primer pair, or the kit in chicken breeding.

[0015] The present application also provides application of the molecular marker, or the primer pair, or the kit in screening of chicken carcass traits.

[0016] The present application discloses the following technical effects:

[0017] The application finds that the CDs region of the EGR1 gene has a SNP site significantly related to chicken carcass traits, provides a new SNP molecular marker for MAS, and finds through test verification that the molecular marker site is significantly related to live weight, half-eviscerated weight, whole-eviscerated weight, wing weight, leg weight and breast muscle rate (p<0.05), wherein the live weight, half-eviscerated weight, whole-eviscerated weight, leg weight, wing weight and breast muscle rate of the GC mutant hybrid genotype individual are significantly higher than those of the CC wild homozygous genotype individual (p<0.05), and the live weight, half-eviscerated weight, whole-eviscerated weight, wing weight, leg weight and breast muscle rate of the GG wild homozygous genotype individual are significantly higher than those of the GC mutant hybrid genotype individual (p<0.05). It can be seen that the molecular marker provided by the application can accurately identify chicken carcass traits, thereby providing a scientific basis for the breeding of chickens. BRIEF DESCRIPTION OF DRAWINGS

[0018] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description only constitute some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0019] Figure 1 EGR1 gene primer pairing position and product length schematic diagram;

[0020] Figure 2 EGR1 gene CDs region SNP site genotyping diagram. DETAILED DESCRIPTION

[0021] The various exemplary embodiments of the present application will now be described in detail, which should not be considered as limiting the present application, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the present application.

[0022] It should be understood that the terms described in the present application are only for describing the specific embodiments, and are not used to limit the present application. In addition, for the numerical range in the present application, it should be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Each smaller range between any stated value or intermediate value in the range, and any other stated value or intermediate value in the range is also included in the present application. The upper limit and the lower limit of these smaller ranges can be independently included or excluded from the range.

[0023] All technical and scientific terms used herein have, unless otherwise defined, the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, preferred methods and materials are described. All publications mentioned in this specification are herein incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. In case of conflict, the content of the present specification will control.

[0024] Various modifications and changes can be made to the specific embodiments described herein without departing from the scope or spirit of the application. Other embodiments will be apparent to those of ordinary skill in the art from the description and examples provided herein. The description and examples are illustrative only.

[0025] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean inclusion, but not limited to, the listed material or step.

[0026] Example 1

[0027] 1. Materials and Methods

[0028] 1.1 Animal samples

[0029] The experimental animals were 356 phenotyped Ephorate chickens of 80 days old. 2 mL of subcutaneous venous blood was collected and stored at -80℃ for DNA extraction. At the same time, the live weight, carcass weight, whole eviscerated weight, half eviscerated weight, wing weight, leg weight, and other carcass traits of the selected population were recorded.

[0030] 1.2 Main reagents

[0031] DNA extraction kit (brand: OMEGA; item number: D3392; Guangzhou Feiyang Biological Engineering Co., Ltd.), 2x Es Taq MasterMix (Dye) (brand: Kangwei; item number: CW0690M; Kangwei Century Biological Technology Co., Ltd.), DNA marker (brand: Novozyme; item number: MD101; Jiangsu Novozyme Biological Technology Co., Ltd.), high-purity low-electrolyte agarose (brand: Qikai; item number: TSJ001; Beijing Qikai Biological Technology Co., Ltd.).

[0032] 2. Experimental methods

[0033] 2.1 Primer design

[0034] According to the sequence of EGR1 gene of red junglefowl (Gallus) published by NCBI (National Center for Biotechnology Information Search database) (Gene ID: 373931), the primer was designed by using the Primer-BLAST tool of NCBI, and the primer synthesis service was provided by Guangzhou Qikexue Biotechnology Co., Ltd. The primer sequence related information is shown in Table 1, and the primer matching position on the EGR1 gene is shown in Table 2. Figure 1

[0035] Table 1 PCR amplification primer sequence

[0036]

[0037] 2.2 Blood sample DNA extraction

[0038] The blood sample DNA was extracted according to the operation manual of the blood sample DNA extraction kit.

[0039] 2.3 PCR amplification of EGR1 gene exon sequence

[0040] The above 356 individual blood sample genomic DNA was used as a template, and the following reaction system was followed: template DNA 2 μL, 2 × Es Taq MasterMix (Dye) 25 μL, upstream primer 2 μL, downstream primer 2 μL, ddH2O 19 μL.

[0041] Reaction procedure: 94℃ pre-denaturation for 2 min; 94℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 30 s, 30 cycles; 72℃ final extension for 3 min; 4℃ storage. The PCR product was purified and sequenced by Shanghai Shengong Biotechnology Engineering Technology Service Co., Ltd.

[0042] 2.4 SNP determination and genotyping

[0043] The sequence peak chart analysis of the sequencing results of the PCR product was performed by using the SeqMan tool of DNAstar software to determine the potential SNP site, and the sequencing data of each sample was compared by using the tool to perform genotyping.

[0044] 2.5 Genotype and carcass trait association analysis

[0045] The SNP site and the genotype of the individual were associated with the carcass trait data, and the SAS 9.4 GLM program package was used for association analysis.

[0046] 3, Results and analysis

[0047] 3.1 PCR amplification and SNP screening of EGR1 gene coding sequence​

[0048] The blood sample DNA of 356 individuals of Ephedra chicken was selected as a template for PCR amplification, and the PCR products were sequenced by Sanger. The peak map after sequencing was analyzed, and a total of 1 SNP site (nucleotide sequence as shown in SEQ ID NO: 3) was detected, wherein 1 site was associated with chicken carcass traits, which was rs313977419C>G, see Figure 2 .

[0049] SEQ ID NO: 3 is:

[0050]

[0051] The above sequence is a SNP site.

[0052] 3.2 Association analysis of SNP site in EGR1 gene coding sequence and carcass traits

[0053] The above 1 SNP site was associated with carcass traits (live weight, half eviscerated weight, whole eviscerated weight, wing weight, leg weight and breast muscle rate).

[0054] As shown in Table 1, the results showed that the rs313977419C>G site was significantly correlated with live weight, carcass weight, half eviscerated weight, whole eviscerated weight, wing weight, leg weight and breast muscle rate (p<0.05). Among them, the breast muscle rate of GC and CC mutant heterozygous genotype individuals was significantly higher than that of GG wild type homozygous genotype individuals (p<0.05), and the live weight, half eviscerated weight, whole eviscerated weight, wing weight and leg weight of GG wild type homozygous genotype individuals were significantly higher than those of CC mutant heterozygous genotype individuals (p<0.05).

[0055] Table 2 Significant association data of SNP site rs313977419 of EGR1 gene and slaughter performance

[0056]

[0057]

[0058] Note: In the same row, the same letter means no significant difference (P>0.05), and different letters mean significant difference (P<0.05)

[0059] The above-described embodiments only describe the preferred modes of the present application, and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those skilled in the art shall fall within the protection scope determined by the claims of the present application.

Claims

1. A method for identifying carcass traits of chicken by using molecular markers, characterized in that, The process includes the steps of amplifying the molecular marker using PCR, analyzing the genotype of the SNP site of the molecular marker, and identifying the carcass traits of the Ma Huang chicken based on the genotype; the carcass traits of the Ma Huang chicken are live weight, semi-eviscerated weight, fully eviscerated weight, wing weight, leg weight, and breast muscle percentage. The nucleotide sequence of the molecular marker is shown in SEQ ID NO:

3. The 736th position of the nucleotide sequence is an SNP site with a G / C mutation. The SNP site contains three genotypes: CC, GC, and GG.

2. The method of claim 1, wherein, The PCR amplification reaction system consisted of: 2 μL template DNA, 25 μL amplification buffer, 2 μL upstream primer, 2 μL downstream primer, and 19 μL ddH2O.

3. The method of claim 1, wherein, The PCR amplification reaction program was as follows: 94℃ pre-denaturation for 2 min; 94℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 30 s, 32 cycles; 72℃ final extension for 3 min; storage at 4℃.

4. The method of claim 1, wherein, When the SNP locus is the GG genotype, the chicken has a higher live weight, semi-eviscerated weight, fully eviscerated weight, wing weight, and leg weight; when the SNP locus is the GC genotype, the chicken has a higher breast muscle percentage.

5. Use of molecular markers in breeding of Gallus gallus domesticus, characterized in that, The nucleotide sequence of the molecular marker is shown in SEQ ID NO:

3. The 736th position of the nucleotide sequence is an SNP site with a G / C mutation. The SNP site has three genotypes: CC, GC, and GG. The traits of the Ma Huang chicken breeding are live weight, semi-eviscerated weight, fully eviscerated weight, wing weight, leg weight, and breast muscle percentage.

6. The use of molecular markers in the selection of carcass traits in chicken, characterized in that, The nucleotide sequence of the molecular marker is shown in SEQ ID NO:

3. The 736th position of the nucleotide sequence is an SNP site with a G / C mutation. The SNP site has three genotypes: CC, GC, and GG. The traits of the Ma Huang chicken breeding are live weight, semi-eviscerated weight, fully eviscerated weight, wing weight, leg weight, and breast muscle percentage.

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