A molecular marker for identifying chicken feed utilization traits based on TIAM2 gene and its identification method and application

Abstract

The present invention relates to a molecular marker for identifying chicken feed utilization traits based on TIAM2 gene, and an identification method and application thereof, wherein the nucleotide sequence of the molecular marker is shown in SEQ ID NO.1, wherein the 299th base of the nucleotide sequence is A or C. The present invention identifies the type of the molecular marker in the chicken genome, selects the chicken feed utilization trait according to the genotype, and establishes a breeding method for early selection of poultry feed utilization, which is simple, rapid, low-cost, does not require special instruments, and is suitable for the needs of molecular marker-assisted breeding experiments.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular markers, and in particular relates to a molecular marker for identifying chicken feed utilization traits based on the TIAM2 gene, and an identification method and application thereof. Background Art

[0002] As the scale of animal husbandry continues to grow, feed costs continue to increase. Therefore, for livestock and poultry farming, feed efficiency traits are economic traits with a greater impact, and feed efficiency needs to be maximized (FATHI MM, AL-HOMIDAN I, EBEID TA, et al. Assessment of Residual Feed Intake and Its Relevant Measurements in Two Varieties of Japanese Quails (Coturnix coturnix japonica) under High Environmental Temperature [J]. 2019, 9: 6.). At present, the two key indicators for evaluating feed efficiency traits at home and abroad are feed conversion rate (FCR) and residual feed intake (RFI) (YUAN J, WANG K, YI G, et al. Genome-wide association studies for feed intake and efficiency in two laying periods of chickens [J]. Genetics Selection Evolution, 2015, 47 (1): 82.). FCR is used to measure the amount of feed consumption required for livestock and poultry to increase one kilogram of body weight during the breeding process, and is a positive selection indicator. RFI is the difference between the expected feed intake and the actual feed intake to maintain growth and development during animal production, and is a negative selection indicator. Selecting animals with low RFI and high FCR not only helps reduce feed costs, but also helps reduce nitrogen waste, thereby minimizing environmental pollution (MOORE SS, MUJIBI FD, SHERMAN E L. Molecular basis for residual feed intake in beef cattle [J]. J Anim Sci, 2009, 87 (14 Suppl): E41-47.).

[0003] T-Lymphoma Invasion And Metastasis-Inducing Protein 2 (TIAM2) can regulate the activity of RHO-like proteins and link extracellular signals to cytoskeletal activity. Secondly, as a GDP dissociation stimulating protein, it stimulates the GDP-GTP exchange activity of RHO-like GTPases and activates them. It can also mediate the specific activation of RAC1 by extracellular laminin signals and promote neurite growth. At present, the research on TIAM2 gene is mainly focused on mice, fish and humans, and there are fewer studies on poultry. The research content mainly focuses on growth and development processes such as muscle development, adipogenesis, and bone development. For example, Song Mingkun found that the TIAM2 gene of meat rabbits plays an important role in the growth and development processes such as muscle development, adipogenesis, and bone development, and is a key candidate gene affecting the growth traits of rabbits (Song Mingkun. Identification and verification of candidate genes affecting rabbit growth traits based on whole genome resequencing [D]. Henan Agricultural University, 2023.). Machado et al. found that the TIAM2 gene is a key candidate gene affecting muscle development in beef cattle (MACHADO PC, BRITO LF, MARTINS R, et al. Genome-Wide Association Analysis Reveals Novel Loci Related with VisualScore Traits in Nellore Cattle Raised in Pasture-Based Systems[J]. ANIMALS, 2022, 12(24): 3526.). However, there is no research on the effects of TIAM2 gene variation and expression on feed conversion efficiency in chickens and its molecular mechanism.

[0004] Huaibei Ma chicken is a small dual-purpose Ma chicken with the advantages of tolerance to roughage, good meat quality, strong adaptability, etc. This application studies the TIAM2 gene variation and finds that the TIAM2 gene variation and expression have a certain effect on the feed conversion rate of chickens. Based on this, a molecular marker is developed, which has important practical significance for studying the influencing factors and molecular mechanisms of the feed conversion rate of chickens, improving the feed efficiency of chickens, and reducing breeding costs. Summary of the invention

[0005] The purpose of the present invention is to provide a molecular marker for identifying chicken feed utilization traits based on the TIAM2 gene, and its identification method and application, to develop single nucleotide polymorphism molecular markers for the candidate gene T lymphoma invasion and metastasis inducing protein 2 (TIAM2 gene) related to the feed utilization traits of chickens, and to provide a new way to achieve early identification of feed utilization traits.

[0006] The present invention achieves the above-mentioned purpose through the following technical solutions:

[0007] A molecular marker for identifying chicken feed utilization traits based on the TIAM2 gene, the nucleotide sequence of the molecular marker is shown in SEQ ID NO.1, the molecular marker is A or C, and the molecular marker is located at the 299th position of the SEQ ID NO.1.

[0008] Application of a molecular marker for identifying feed utilization traits of chickens based on the TIAM2 gene in identifying feed utilization traits of chickens.

[0009] A method for identifying chicken feed utilization traits using molecular markers comprises the following steps:

[0010] (1) Extracting total DNA from chicken wing venous blood;

[0011] (2) designing specific amplification primers based on the sequence of the site where the molecular marker is located and its upstream and downstream bases as the target sequence, using the total DNA as a template, and performing PCR amplification using the specific amplification primers to obtain an amplified product;

[0012] (3) performing genotyping detection and sequencing on the amplified product to obtain the molecular marker type of the chicken to be tested;

[0013] (4) Determine the chicken feed utilization trait according to the molecular marker type: if the chicken molecular marker type to be tested is AC type, the chicken feed utilization trait is poor; if the chicken molecular marker type to be tested is CC type, the chicken feed utilization trait is the best; if the chicken molecular marker type to be tested is AA type, the chicken feed utilization trait is medium.

[0014] As a further optimization scheme of the present invention, the sequence of the specific amplification primer is:

[0015] SEQ ID NO.2: Forward primer: ATCCAGCAATGGGAACAG;

[0016] SEQ ID NO.3: Reverse primer: AGGAAGAGGCAGAAGCAA.

[0017] As a further optimization scheme of the present invention, the genotyping detection method is to obtain an enzyme cleavage product by enzymatic cleavage of the amplified product, detect the enzyme cleavage product by agarose gel electrophoresis, and perform genotyping based on the image. If the enzyme cleavage product is one band, it is AA type; if the enzyme cleavage product is two bands, it is CC type; if the enzyme cleavage product is three bands, it is AC type.

[0018] As a further optimization scheme of the present invention, if the chicken molecular marker type to be tested is AC type, the chicken feed utilization trait is poor; if the chicken molecular marker type to be tested is CC type, the chicken feed utilization trait is the best; if the chicken molecular marker type to be tested is AA type, the chicken feed utilization trait is medium.

[0019] As a further optimization scheme of the present invention, HaeIII restriction endonuclease is used for enzyme digestion.

[0020] As a further optimization scheme of the present invention, the enzyme cleavage products were detected by agarose gel electrophoresis at a concentration of 1.5%-2.0%.

[0021] The present invention provides the following beneficial effects:

[0022] The present invention develops a molecular marker related to chicken feed utilization based on the TIAM2 gene. By identifying the type of the molecular marker present in the chicken genome, the feed utilization trait of the chicken is selected according to the genotype, and a breeding method for early selection of poultry feed utilization is established. The method is simple, rapid, low-cost, does not require special instruments, and is suitable for the needs of molecular marker-assisted breeding experiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Agarose gel electrophoresis of PCR amplification products of some samples;

[0024] Figure 2 It is the agarose gel electrophoresis diagram of the digestion products obtained by digestion of PCR amplification products of some samples;

[0025] Figure 3 The sequencing results for genotype verification of the A105910C site in the chicken TIAM2 gene (site 299 in SEQ ID NO.1). DETAILED DESCRIPTION

[0026] The present application is further described in detail below in conjunction with the accompanying drawings. It is necessary to point out here that the following specific implementation methods are only used to further illustrate the present application and cannot be understood as limiting the scope of protection of the present application. Technical personnel in this field can make some non-essential improvements and adjustments to the present application based on the above application content.

[0027] 1. Materials

[0028] The methods used in this example are conventional methods known to those skilled in the art unless otherwise specified, and the reagents and other materials used are commercially available products unless otherwise specified.

[0029] 2. Methods

[0030] 2.1 Primer design

[0031] According to the DNA sequence of the TIAM2 gene disclosed in the chicken genome database, the sequence consisting of the A105910C site of the chicken TIAM2 gene and its upstream and downstream bases was used as a template (the template sequence is shown in SEQ ID NO.1, and the A105910C site corresponds to the 299th site of SEQ ID NO.1), and the specific amplification primers were designed. The sequences of the specific amplification primers are shown below:

[0032] SEQ ID NO.2: Forward primer: ATCCAGCAATGGGAACAG;

[0033] SEQ ID NO.3: Reverse primer: AGGAAGAGGCAGAAGCAA.

[0034] The length of the amplifiable region of the primer is 332 bp, and the amplified sequence is shown in SEQ ID NO.4, which contains the molecular marker of the A / C mutation at the 299th position in SEQ ID NO.1 (position A105910C).

[0035] 2.2 Extraction of blood genomic DNA

[0036] 474 Huaibei Ma chickens were selected, blood was collected from the wing vein and stored in EDTA anticoagulant tubes, and the blood genomic DNA extraction kit produced by Tiangen Biotechnology Co., Ltd. was used to extract the sample DNA. The extraction steps were carried out according to the kit instructions.

[0037] 2.3 PCR amplification

[0038] Using 2×Hieff PCR Master Mix (With Dye) produced by Yisheng Biotechnology Co., Ltd., PCR amplification of the target fragment of the TIAM2 gene was performed using synthetic specific primers. The PCR amplification system is shown in Table 1:

[0039] Table 1 PCR amplification system

[0040] Components Dosage DNA template 1μL Forward primer 1μL Reveres primer 1μL Mix 12.5μL <![CDATA[ddH2O]]> 9.5μL total 25μL

[0041] The PCR reaction conditions were as follows: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 sec, annealing for 30 sec, extension at 72°C for 30 sec, and final extension at 72°C for 10 min, wherein the denaturation to extension phases were cycled 35 times in total.

[0042] 2.4 PCR amplification product detection and sequencing

[0043] The PCR amplification products were detected by 1.5% agarose gel electrophoresis. Figure 1As shown, after imaging with a gel imager, a band of approximately 332 bp in length was obtained, which was consistent with the predicted length, indicating that the target fragment was specifically amplified. The PCR product was sent to Beijing Qingke Biotechnology Co., Ltd. (Nanjing) for sequencing, and the sequence was shown in SEQ ID NO.4, which was consistent with the predicted result.

[0044] 2.5 Genotyping

[0045] 2.5.1 The enzyme digestion system shown in Table 2 was prepared. The digestion conditions were 37°C for 12 hours. The PCR amplification product was digested with HaeIII restriction endonuclease from Thermo Fisher Scientific.

[0046] Table 2 Enzyme digestion system

[0047] Components Dosage PCR amplification products 6μL HaeIII 0.2μL (10U / μL) buffer 1μL <![CDATA[ddH2O]]> 2.8μL total 10μL

[0048] 2.5.2 Using 1.5% agarose gel electrophoresis, the following Figure 2 The results shown (partial results); if the enzyme cleavage product is 1 band, it is AA type; if the enzyme cleavage product is 2 bands, it is CC type; if the enzyme cleavage product is 3 bands, it is AC type.

[0049] 2.6 Enzyme Digestion and Sequencing Verification

[0050] The agarose gel electrophoresis diagram of gene restriction typing was statistically analyzed to obtain three typings: AA, CC, and AC. One individual was selected for each of the three typings for sequencing comparison. The sequencing comparison diagram is shown in Figure 3 As shown, the sequencing results revealed that the molecular marker site had mutated from A to C. The arrow indicates the mutation location, which is consistent with the enzyme typing results.

[0051] 2.7 Effect Verification

[0052] In order to determine the association between the A / C polymorphism of the molecular marker A105910C site of the chicken TIAM2 gene and important phenotypic traits of chickens, 474 Huaibei Ma chickens in 2.2 were used as experimental materials, and the average daily feed intake (ADFI), average daily weight gain (ADG), metabolic body weight (MBW) at 70 to 120 days of age were statistically analyzed. 0.75 ), feed conversion rate (FCR), residual feed intake (RFI), 474 Huaibei Ma chickens were genotyped using the 2.5 genotyping method, and the results are shown in Table 3:

[0053] Table 3 Genotype detection results of individuals with different phenotypes

[0054]

[0055]

[0056] Experimental conclusion: The chi-square test results showed that the genotype of the experimental chicken population was in Hardy-Weinberg equilibrium (P>0.05).

[0057] 2.8 Statistical analysis

[0058] The association between the three genotypes and the chicken feed utilization traits was analyzed using the one-way analysis of variance program in SPSS software. The results of the association analysis between different genotypes and each trait are shown in Table 4:

[0059] Table 4 Association analysis between chicken TIAM2 genotype and chicken feed utilization traits

[0060]

[0061] Note: Different lowercase letters in the same row indicate significant differences (P<0.05), and different uppercase letters in the same row indicate extremely significant differences (P<0.01).

[0062] Experimental conclusion: According to Table 4, at the A105910C mutation site of the TIAM2 gene, the ADFI of AA and AC types was significantly greater than that of CC type (P < 0.01); the RFI of AC type was significantly greater than that of CC type (P < 0.05); the MBW of AA and AC types was significantly greater than that of CC type (P < 0.05). 0.75 It was significantly greater than the CC type (P < 0.05). There was no significant difference in ADG and FCR among the three genotypes. It can be concluded that the feed utilization trait of the AC genotype individuals was poor, the feed utilization trait of the AA genotype individuals was medium, and the feed utilization trait of the CC genotype individuals was the best.

[0063] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. An application of a molecular marker based on the TIAM2 gene in identifying the feed utilization trait of chickens, characterized in that: The nucleotide sequence of the molecular marker is shown in SEQ ID NO.1, wherein the 299th base of the nucleotide sequence is A or C; If the chicken molecular marker type to be tested is AC type, the chicken feed utilization trait is poor; if the chicken molecular marker type to be tested is CC type, the chicken feed utilization trait is the best; if the chicken molecular marker type to be tested is AA type, the chicken feed utilization trait is medium; The chicken to be tested is Huaibei Ma chicken, and the feed utilization traits are average daily feed intake, metabolic body weight, and residual feed intake.

2. A method for identifying chicken feed utilization traits based on molecular markers of the TIAM2 gene, characterized in that: The following steps are involved: (1) Extract total DNA from venous blood of Huaibei hemp chicken wings; (2) designing specific amplification primers based on the sequence of the site where the molecular marker is located and its upstream and downstream bases as the target sequence, using the total DNA as a template, and performing PCR amplification using the specific amplification primers to obtain an amplified product; The nucleotide sequence of the molecular marker is shown in SEQ ID NO.1, wherein the 299th base of the nucleotide sequence is A or C; (3) performing genotyping detection and sequencing on the amplified product to obtain the molecular marker type of the chicken to be tested; (4) Determining the feed utilization trait of the chicken according to the molecular marker type: if the molecular marker type of the chicken to be tested is AC type, the feed utilization trait of the chicken is poor; if the molecular marker type of the chicken to be tested is CC type, the feed utilization trait of the chicken is the best; if the molecular marker type of the chicken to be tested is AA type, the feed utilization trait of the chicken is medium; The feed utilization traits are average daily feed intake, metabolic body weight, and residual feed intake.

3. The method for identifying chicken feed utilization traits based on molecular markers of TIAM2 gene according to claim 2, characterized in that: The sequence of the specific amplification primer is: SEQ ID NO.2: Forward primer: ATCCAGCAATGGGAACAG; SEQ ID NO.3: Reverse primer: AGGAAGAGGCAGAAGCAA.

4. The method for identifying chicken feed utilization traits based on molecular markers of the TIAM2 gene according to claim 3, characterized in that: The genotyping detection method is to obtain an enzyme digestion product by enzymatic digestion of the amplified product, detect the enzyme digestion product by agarose gel electrophoresis, and perform genotyping according to the image. If the enzyme digestion product is one band, it is AA type; if the enzyme digestion product is two bands, it is CC type; if the enzyme digestion product is three bands, it is AC type.

5. The method for identifying chicken feed utilization traits based on molecular markers of TIAM2 gene according to claim 4, characterized in that: The fragments were digested with HaeIII restriction endonuclease.

6. The method for identifying chicken feed utilization traits based on molecular markers of TIAM2 gene according to claim 4, characterized in that: The enzyme digestion products were detected by 1.5%-2.0% agarose gel electrophoresis.

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